Compound distribution in microfluidic devices

ABSTRACT

The present invention is related to the field of microfluidics and compound distribution within microfluidic devices and their associated systems. In one embodiment, present invention aims to solve the problem of molecule and compound absorbency into the materials making up laboratory equipment, microfluidic devices and their related infrastructure, without unduly restricting gas transport within microfluidic devices.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of, and claims priority to,co-pending PCT Patent Application Serial No. PCT/US2020/039830, filedJun. 26, 2020, which claims priority to Provisional Application Ser. No.62/867,543 filed on Jun. 27, 2019 now expired, the contents of which areincorporated herein in their entirety.

FIELD OF INVENTION

The present invention is related to the field of microfluidic cellculture systems. A variety of microfluidic devices and a perfusionmanifold assembly are contemplated that limit small-molecule absorptioninto their material makeup. An absorption kit or compound distributionkit is also contemplated to characterize the movement of a compoundwithin an experimental or clinical system. A gas exchanger is alsocontemplated to control the rate of gas flow from one area to another,such as from the ambient environment to the interior of a microfluidicdevice.

BACKGROUND

New microfluidics technology offers useful experimental tools forstudying cells ex vivo. Compared with conventional culture systems, amicrofluidic device can provide a more physiologically relevant cellularenvironment, by generating fluid flows which can maintain a moreconstant and soluble microenvironment, such as described by Breslauer etal. in the publication “Microfluidics-based systems biology.”Two-dimensional (2D) monolayer cell culture systems have been used formany years in biological research. The most common cell culture platformis the two-dimensional (2D) monolayer cell culture in petri dishes orflasks. Although such 2D in vitro models are less expensive than animalmodels and are conducive to systematic, and reproducible quantitativestudies of cell physiology (e.g., in drug discovery and development),the physiological relevance of the in vitro systems to the in vivocondition is often questionable and the results from such studies oftenlack validity. It has now been widely accepted that three-dimensional(3D) cell culture matrix promotes many biological relevant functions notobserved in 2D monolayer cell culture. Said another way, 2D cell culturesystems do not accurately recapitulate the structure, function, andphysiology of living tissues in vivo.

U.S. Pat. No. 8,647,861 describes microfluidic “organ-on-chip” devicescomprising living cells on membranes in microchannels exposed to culturefluid at a flow rate. In contrast to static 2D culture, microchannelsallow the perfusion of cell culture medium throughout the cell cultureduring in vitro studies and as such offer a more in vivo-like physicalenvironment. In simple terms, an inlet port allows injection of cellculture medium into a cell-laden microfluidic channel or chamber, thusdelivering nutrients and oxygen to cells. An outlet port then permitsthe exit of remaining medium as well as harmful metabolic by-products.

U.S. patent application Ser. No. 15/248,690 describes a perfusionmanifold assembly that retains one or more microfluidic devices, such as“organ-on-chips”, that comprise cells that mimic at least one functionof an organ in the body, and allow the perfusion and optionally themechanical actuation of said microfluidic devices, optionally withouttubing. The perfusion manifold assembly interacts with a culture modulethat allows the perfusion and optionally mechanical actuation of one ormore microfluidic devices, such as “organ-on-chip” devices. The culturemodule comprises a pressure manifold that allows for perfusion ofmicrofluidic devices, such as the “organ-on-chip” devices.

The materials oftentimes used to fabricate these microfluidic devices or“organ-on-chip” devices, and their relating infrastructure, such as theperfusion manifold assembly, are oftentimes prone to erroneousdistribution of compounds and absorption of small molecule compoundsthey are designed to non-absorbingly interface with. While the materialsused to construct microfluidic devices and their infrastructure, such aspolydimethylsiloxane (PDMS) and the styrenic block copolymer (SEBS)manufactured by Kraton, tend to be widely available, inexpensive andamendable to microfluidic device fabrication, they inhibit particularvarieties of research they otherwise would be ideal for, such as thedrug discovery and development, etc. For the purposes of drug discoveryand development, microfluidic device absorbency can lead to variabilityin drug exposure to subjects in the microfluidic device, such as patientcells, animal cells, microbial cells, small organisms, etc. In order toenable therapeutic prediction, there needs to be a high confidence inthe concentration of a compound that specimen is exposed to.

Despite absorbency issues, materials used to fabricate microfluidicdevices, such as PDMS, also tend to promote the transport of gassesvital in supporting the viability and function of the subjects insidethe microfluidic device. In particular, oxygen from the outsideenvironment is allowed to permeate/pass to the interior of themicrofluidic device, supplying the cells with oxygen needed forrespiration. Similarly, carbon dioxide, which is released duringcellular respiration, is allowed to pass through the material frominside the microfluidic device to the external environment; this preventthis waste product from building-up and becoming toxic to the cells.Non-absorbing or low-absorbing materials, such as many hard plastics,tend to hinder the transport of these gasses into and out ofmicrofluidic devices. What is needed is a solution to decouple gaspermeability, which is needed to support biological viability andfunction, from absorbency, which limits the applications of thesedevices in the research of pharmaceutical, in the materials used inmicrofluidic devices.

SUMMARY OF THE INVENTION

The present invention is related to the field of microfluidics. Thepresent invention is related to compound distribution withinmicrofluidic devices and their associated systems. In one embodiment,present invention aims to solve the problem of molecule and compoundabsorbency into the materials making up laboratory equipment,microfluidic devices and their related infrastructure, withoutrestricted gas transport within microfluidic devices. Compounds, such aspharmaceuticals and chemicals, can absorb into, bind to, or poorlydistribute within various components of in vivo and in vitroexperimental setups, with frequent offenders including infusion tubing,syringes, tissue-culture labware, and pipette tips. Such absorptionbinding, and poor distribution often goes unnoticed, therebycontributing to variability and skewing quantitative experimentalresults (e.g. dose-response curves). Microfluidic devices are noexception. Microfluidic devices fabricated from entirely gas-permeablematerials tend to absorb small molecule compounds disrupting data,thereby confounding the data collected using those microfluidic devices.For example, the material polydimethylsiloxane (PDMS) is highlyabsorptive, which can negatively affect experiments, especially if thatabsorption is not understood. Absorption of small molecules into thebulk material making up microfluidic devices decreases the concentrationof those small molecules that may come into contact with specimen withinmicrofluidic devices, such as cells and small organisms.

It is estimated that small-molecule compounds fall into one of threecategories of absorption, (1) compounds that do not absorb at detectablelevels, (2) compounds that somewhat absorb at levels that can bedetected, and (3) compounds that highly or completely absorb. It isestimated that about 40% of small-molecule compounds do not absorb,falling in the first category. It is estimated that about 40% ofsmall-molecule compounds somewhat absorb, falling into the secondcategory. It is estimated that about 20% of small-molecule compoundscompletely absorb, falling into the third, most difficult, category. Assuch, it is estimated that approximately 60% of small-molecule compoundscould be potentially problematic not only during scientificexperimentation, but also during live patient dosing.

Small molecules may also be known as xenobiotics. Xeno means foreign.Xenobiotics tend to be what is thought of classically as chemicals,which do not typically occur in the human body. Xenobiotics tend to besmaller molecules compared to biologics. Biologics, such as proteins andantibodies, naturally occur in the human body and tend to be largermolecules as compared to xenobiotics. In practice, most xenobiotics areunder 1 kDa in molecular weight. For the purpose of the presentinvention a molecule under 1 kDa in molecular weight may be considered asmall molecule.

Some materials are more prone to material absorption than others.Polymers can generally be seen as rigid or elastomeric. Polymers may begauged as rigid or elastomeric based on their Young's Moduli, also knownas flexural modulus, also known as modulus of elasticity. In practice,any polymer with a modulus of elasticity over 0.1 GPa is consideredeffectively rigid, or non-flexible, certainly for the purposes ofmicrofluidic device fabrication. Rigid polymers may fall in the range of0.1 GPa to 150 GPa. Metals usually have a modulus of elasticity value ofat least 30 GPa or greater. For example, aluminum can have a modulus ofelasticity value up to about 69 GPa. In some embodiments, the rigidityor flexibility of materials and/or the biocompatible material can bedetermined by the material hardness. For example, hardness of a materialcan be typically measured by its resistance to indentation under astatic load. The most commonly used measures are the Shore hardness andRockwell hardness. Both are empirical relative measures. The Shorehardness is a measure often used as a proxy for flexural modulus ofelastomers. The Shore A scale is typically used for softer elastomerswhile Shore scale D is used for harder elastomers or softer rigidthermoplastic materials. By way of example only, rigid but softerthermoplastic materials such as polypropylenes can have typical valuesbetween 75 and 85 on the Shore D scale. Harder rigid thermoplasticmaterials such as acrylic can be usually characterized on Rockwell Mscale. For example, Rockwell M value of acrylic can be 85-105,polycarbonate 72, polystyrene 68-70, and polysulfone 70.

Few materials fall between being flexible or elastomeric and rigid. Saidanother way, when looking at moduli of elasticity, very few materialsfall in a range where it would be ambiguous if they are elastomeric orrigid. Materials that fall may be considered between flexible orelastomeric and rigid, including polytetrafluorethylene (PTFE) orTeflon, and fluorinated ethylene propylene (FEP), have been tested andhave been found to be absorbing. Therefore, only rigid materials maygenerally be considered low-absorbing. Another class of flexiblepolymers are rubbers, which are different from plastics. Rubbers tend tobe flexible as well. Rubbers include natural rubber and liquid siliconerubber. However, it has been found that rubbers generally tend to absorbsmall molecules as well.

Following an extensive look into both rigid and elastomeric polymers ithas generally been seen that rigid polymers are low-absorbing, whileelastomeric polymers are prone to absorption of small molecules. Withoutbeing bound by theory, molecules or monomers within elastomeric polymersgenerally have higher mobility than molecules within rigid polymers.Polymers are made up of monomers, or molecules that come togetherthrough polymerization to form a polymer. As the molecules or monomerswithin rigid polymers are not as mobile, it is more difficult formolecules to absorb or diffuse into rigid polymers. Conversely, as themolecules or monomers within elastomeric polymers as more mobile, it isless difficult for molecules to absorb or diffuse into elastomericpolymers. Therefore, when using elastomeric polymers for their otherbeneficial properties, such as their ability to stretch, one must relymore heavily on chemical rather than physical parameters to stopabsorption or diffusion of other molecules into the polymer. Chemicalparameters may not only entail coatings, but also the chemicalproperties of the polymer, such as dispersion, polarity, and hydrogenbonding.

Hansen solubility parameters developed by Charles M. Hansen predicted ifone material will dissolve in another. For the uses herein, the Hansensolubility parameters are helpful in predicting the solubility of amolecule into a solid. The three Hansen solubility parameters aredispersion (δD), polarity (δP), and hydrogen bonding (δH). Dispersionforces, also known as Van der Waals forces, are the distance-dependentinteractions between molecules, such as attraction and repulsion.Polarity is the uneven partial charge distribution between various atomsin a compound. Hydrogen bonding are the attractive forces betweenhydrogen atoms covalently bonded to very electronegative atoms, such asnitrogen, oxygen and fluorine and another very electronegative atom.Materials with similar Hansen solubility parameters are more likely todissolve into each other. The following equation may be used todetermine the Hansen solubility parameter of a material:

(Ra)²=4(δ_(D2)−δ_(D1))²+(δ_(P2)−δ_(P1))²+(δ_(H2)−δ_(H1))²

Where Ra is the is the distance between Hansen parameters in Hansenspace. The closer the Hansen parameters the more likely the two materialare to dissolve into each other. δ_(D1) is the dispersion force of thefirst material. δ_(P1) is the polarity of the first material. δ_(H1) isthe hydrogen bonding of the first material. δ_(D2) is the dispersionforce of the second material. δ_(P2) is the polarity of the secondmaterial. δ_(H2) is the hydrogen bonding of the second material.

Following a detailed and mathematical look into both elastomeric andrigid polymers, there has not been a polymer found that may make up thebody of a multi-purpose microfluidic device. It is desired that themicrofluidic device be low-absorbing, able to stretch in order to betteremulate in vivo physiology, as well as biocompatible. As stated before,rigid polymers tend not to absorb. Elastomeric polymers are flexible. Assuch, there has not yet been found any elastomeric nor rigid polymersthat, in their own right, both resist molecule absorption and alsostretch. As such, as strategic combination of elastomeric and rigidpolymers may be able to be used, in one embodiment, to fabricate aflexible, low-absorbing microfluidic device.

Absorption into the body of the microfluidic device negatively impactsexperimental readouts, such as cellular metabolism. A challenge whenusing microfluidic devices is understanding and quantifying the rate ofmetabolism and the resulting metabolite produced when cells are incontact with candidate compounds. In order to deduce intrinsic clearanceof drugs in the case of, say, liver cells, the metabolism, or loss ofthe parent compound, is often measured. Intrinsic clearance is theability of the liver to remove drug in the absence of limitations, suchas flow or binding to cells or proteins in the blood. Low rates ofmetabolism can make it difficult to detect loss of the parent compound,even if the microfluidic device is non-absorbing. Most importantly,absorption can make it impossible to deduce whether compound loss shouldbe attributed to metabolism or absorption. In simpler terms, if cellsconsume low concentrations of a parent compound, it is difficult toquantify that loss. A practical level of sensitivity of detection in anLC/MS instrument is ±25%. As such, a decrease in the concentration ofthe parent compound needs to be at least 25% and ideally much more inorder to effectively quantify metabolism. Quantifying metabolism is mademore difficult by absorption of the parent compound. If absorption intothe material, such as PDMS, is significant, then the observed apparentrate of metabolism (if all of compound loss is attributed to metabolism)will over-estimate actual cell-mediated metabolism as the decrease incompound concentration will be incorrectly attributed to metabolism. Insome cases, all of the parent compound could be depleted by thematerial. In this case, absorption will prevent even an estimation ofthe upper possible rate of metabolism, since there will be no data toanalyze as all of the compound has been lost. Material absorption can becomputationally modeled and accounted for given information on thematerial-compound properties, such as the rate and extent of absorptionin the material, experimental parameters, such as dosing concentrationand flow rate, and microfluidic device geometry as long as all of theparent compound is not being depleted by the material. Computationalmodeling, however, requires extensive studies to characterize thecompound-material interaction as well as computationally expensivemodels of the system to “subtract out” the contribution of materialabsorption to loss or disappearance of compound. If compound loss iscomplete, these models cannot account for the contribution ofabsorption, as compound loss is complete.

The present invention is made up of multiple unique embodiments. Oneembodiment of the present invention is a method for using a rigidmicrofluidic device with a high flow rate (e.g. greater than 40 μL/hr)in order to introduce important gases into the channels of themicrofluidic device. Another embodiment of the present invention is amethod for using a rigid microfluidic device with a recirculated fluid(in one embodiment, flowing at a high flow rate), in order to bothreduce the volume of fluid being used and also maintain the importantchemical and biological material within the fluid within the fluid foruse within the microfluidic device, such as cell signals. Anotherembodiment of the present invention is a method for using a rigidmicrofluidic device with a reciprocated fluid (in one embodiment,flowing at a high flow rate), in order simplify the experimental setup,reduce the volume of fluid being used, and also maintain the importantchemical and biological material within the fluid for use within themicrofluidic device, such as cell signals. Another embodiment of thepresent invention is a rigid microfluidic device comprising a gasexchanger in order to introduce gases into the channels of themicrofluidic device. Another embodiment of the present invention is agas exchanger, the gas exchanger made up of a rigid polymer comprisingpores, the pores filled with an elastomeric polymer. Another embodimentof the present invention is an elastomeric microfluidic devicecomprising a gas exchanger and rigid polymer masks. Another embodimentof the present invention is a microfluidic device, also known as a “halochip,” comprising gas channels along the perimeter of the workingchannels of the microfluidic device in order to encourage gas flowbetween the gas and working channels. Another embodiment of the presentinvention is a rigid microfluidic device comprising elastomeric channelwalls and an elastomeric membrane between a first channel and a secondchannel. Another embodiment of the present invention is a rigidmicrofluidic device comprising an elastomeric membrane between a firstchannel and a second channel, such that the elastomeric channelstretches when differential pressure is applied across the elastomericmembrane. Another embodiment of the present invention is a low-absorbingperfusion manifold assembly representing fluidic infrastructure aroundthe microfluidic device. Another embodiment of the present invention isa compound distribution kit used to determine compound absorption intomaterials that make up experimental and clinical systems. Themicrofluidic devices and the low-absorbing perfusion manifold assemblypresented herein aim to minimize small molecule absorption, while themicrofluidic devices also aim to allow ambient gases to accessexperimental regions of the devices, such as microfluidic channelscontaining living cells. The microfluidic devices, the methods to usethem, and the low-absorbing perfusion manifold assembly were alldesigned following the surprising discovery that many elastomericmaterials absorb small-molecules, such as those found in many compounds(drugs, chemicals, cosmetics etc.) U.S. Pat. No. 8,647,861 describes amicrofluidic device, or organomimetic device, or device capable ofmimicking the functionality of an organ, comprising: a body having acentral microchannel therein; and an at least partially porous membraneconfigured to form a first microchannel and a second microchannel,wherein a first fluid may be applied through the first microchannel anda second fluid may be applied through the second microchannel, themembrane may optionally be coated with at least one attachment moleculethat supports adhesion of a plurality of living cells wherein the porousmembrane is at least partially flexible. In one embodiment, the devicefurther comprising: a first operating channel separated the first andsecond central microchannels by a first microchannel wall, wherein themembrane is fixed to the first chamber microchannel wall; and whereinapplying a pressure to the first operating channel causes the membraneto flex in a first desired direction to expand or contract along theplane within the first and second central microchannels. Themicrofluidic device of U.S. Pat. No. 8,647,861 may be fabricated out ofelastomeric polymers, such as PDMS.

One embodiment of the present invention is to apply rigid polymer thinfilms or masks. Thin, rigid polymer films or masks serve to limitgaseous transport into the body of the microfluidic device from theambient environment. One use of the microfluidic devices in U.S. Pat.No. 8,647,861 are to study seeded cells, such as certain varieties ofgut cells that are native to low-oxygen environments. Many elastomericpolymers are highly permeable to gas transport, so much so that somevarieties of cells express higher levels of viability with limited gastransport into the microfluidic device housing them. As such, thin filmsor masks fabricated from rigid polymers may be put into contact with theoutside surfaces of the microfluidic devices fabricated from elastomericpolymers in order to limit the transport of gases into the bodies of themicrofluidic devices. Rigid polymers include, but are not limited topolyethylene terephthalate (PET), cyclic olefin copolymer (COP),polytetrafluorethylene, polypropylene, polyethylene terephthalate andpolyvinyl chloride, acrylic, polystyrene, polycarbonate, glass, epoxyfiberglass, ceramic and metal. A method is contemplated providing anelastomeric microfluidic device comprising outside surfaces and one ormore thin films of rigid polymer, and putting said one or more thinfilms of rigid polymer in contact with said outside surfaces. A methodis contemplated providing an elastomeric microfluidic device comprisingthin film of rigid polymer and a first channel and a second channelseparated by a membrane, and placing said thin film of rigid polymer incontact with an outer surface of said elastomeric microfluidic device. Afluidic device is contemplated comprising an elastomeric body comprisingone or more channels separated by a membrane, outer surfaces, and thinfilms of rigid polymer in contact with at least one of said outersurfaces. A system is contemplated comprising a microfluidic devicecomprising a first channel and a second channel separated by a membrane,and one or more outer surfaces, said outer surfaces in contact with oneor more thin films of rigid polymer. Said thin films may be consideredmasks. For purposes herein, a mask may be considered a layer of polymerfor either restricting or allowing gas or molecule transport.

In one embodiment, the present invention contemplates a fluidic device,said fluidic device comprising (i) a plurality of outer sides, (ii) afirst channel disposed within said body, (iii) a second channel disposedwithin said body, (iv) a membrane disposed between said first channeland second channel, and (iv) one or more gas-impermeable maskscontacting one or more of said plurality of outer sides.

In one embodiment, a method of controlling gas transport iscontemplated, comprising: a) providing a substantially gas-permeablemicrofluidic device comprising i) a plurality of outer sides and ii)living cells in an inner channel or chamber, said microfluidic devicecomprising an elastomeric polymer having a modulus of elasticity below0.1 GPa; and one or more gas-impermeable masks having a modulus ofelasticity between 0.1 and 150 GPa; b) contacting at least one of saidplurality of outer sides with a gas-impermeable mask; and c) introducingnon-oxygenated culture media into said channel or chamber at a flowrate.

In one embodiment, a fluidic device is contemplated, said fluidic devicecomprising (i) a plurality of outer sides, (ii) a first channel disposedwithin said body, (iii) a second channel disposed within said body, (iv)a membrane disposed between said first channel and second channel, (iv)a gas exchanger contacting at least one of said first channel and secondchannel, and (v) one or more gas-impermeable masks contacting one ormore of said plurality of outer sides.

In one embodiment, the present invention contemplates a method ofcontrolling gas transport, comprising: a) providing a substantiallygas-permeable microfluidic device comprising i) a plurality of outersides, ii) a gas exchanger and iii) living cells in an inner channel orchamber, said device comprising an elastomeric polymer having a modulusof elasticity below 0.1 GPa; b) adding a substantially gas-impermeablemask to at least one of said plurality of outer sides without maskingthe gas exchanger; and c) introducing non-oxygenated culture media intosaid channel or chamber at a flow rate, wherein the rate of gastransport to said living cells is controlled by said gas exchanger. Inone embodiment, said substantially gas-impermeable mask comprises apolyethylene terephthalate (PET) film.

One embodiment of the invention presented herein is an improvement onthe microfluidic device presented in U.S. Pat. No. 8,647,861, followingthe discovery that the materials most commonly used to fabricate themicrofluidic devices in U.S. Pat. No. 8,647,861 (e.g. PDMS) have thepotential to be highly absorptive to xenobiotics and small molecules.

In one embodiment, a microfluidic device is contemplated comprising aplurality of outer sides comprising substantially gas-permeable polymerhaving a modulus of elasticity below 0.1 GPa, and a substantiallygas-impermeable mask attached to at least one of said plurality of outersides. In one embodiment, said substantially gas-impermeable maskcomprises a polyethylene terephthalate (PET) film.

One embodiment of the present invention is to apply coatings. Themicrofluidic device, the perfusion manifold assembly, and/or theircomponents may be coated. Coatings may be applied in a variety ofmethods, such as through films, brushed on, spray coating, spin coating,vapor deposition, rolled on, plating, dip coating, etc. The coating maybe all-over, completely covering the substrate, or the coating may onlycover parts or portions of the substrate.

Coatings may be applied to change the surface properties of thesubstrate, such as absorption resistance, gas resistance, wettability,adhesion, corrosion resistance, wettability, electrical conductivity,etc. The coatings may be applied as a specified thickness. Coatings maybe applied as liquids, gases or solids.

To overcome the problem of absorption into the body of microfluidicdevices such as those disclosed in U.S. Pat. No. 8,647,861, microfluidicdevices were redesigned to be built from rigid materials, such as, butnot limited to, glass, cyclic olefin copolymer (COP), etc. These rigidmicrofluidic devices are detailed in PCT/US2014/071570. In oneembodiment, the invention presented herein comprises a low-absorbing,gas-impermeable microfluidic device. In simple terms, a microfluidicdevice that would limit small-molecule absorption, as well as gas flow,into the bulk of the microfluidic device. In one embodiment, thislow-absorbing, gas-impermeable microfluidic device is fabricated from arigid polymer. In one embodiment, the invention presented hereincomprises a rigid microfluidic device comprising a first channel and asecond channel separated by a membrane. In one embodiment, thelow-absorbing, gas-impermeable microfluidic device comprises a solidsubstrate comprising one or more microfluidic channels. While designeden route to a low-absorbing, gas-permeable microfluidic device, thelow-absorbing, gas-impermeable microfluidic device is a unique inventionwith a variety of useful applications. In one embodiment, the rigid orlow-absorbing, gas-impermeable microfluidic device comprises a pluralityof microfluidic channels. In an exemplary embodiment, the rigid orlow-absorbing, gas-impermeable microfluidic device comprises: a) asubstrate comprising a one or more microfluidic channels, and b) aporous membrane separating said one or more microfluidic channel intoone or more first chambers and second chambers. It is not intended thatthe microfluidic device be limited by substrate, membrane, chamber orchannel configuration. In one embodiment, said first and second chambersare oriented vertically. In one embodiment, said first and secondchambers are oriented horizontally. Said first and second chambers mayalso be referred to as channels. Said first and second chambers, iforiented horizontally, may be referred to as top and bottom chambers orchannels. A first fluid may be applied through said first chamber. Asecond fluid may be applied through said second chamber.

Low-absorbing, gas-impermeable microfluidic devices may be fabricatedusing rigid or low-absorbing, gas-impermeable materials such as, but notlimited to, glass, cyclic olefin copolymer (COP), etc. Low-absorbing,gas-impermeable microfluidic devices may be considered advantageous tohigh-absorbing, gas-permeable microfluidic devices, such as thosefabricated from elastomeric materials including PDMS, as they aregenerally resistant to absorption of small molecules, unlikehigh-absorbing, gas-permeable microfluidic devices. Said another way,rigid microfluidic devices may be considered advantageous to elastomericmicrofluidic devices, as rigid microfluidic devices may be consideredimpermeable to absorption of small molecules or xenobiotics. In someembodiments, the rigid materials can reduce absorption of molecules byat least about 10% or more, including, e.g., at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90% or more,as compared to the extent of molecule absorption into PDMS.

In one embodiment, a microfluidic device is contemplated comprising aplurality of outer sides comprising substantially gas-impermeablepolymer having a modulus of elasticity between 0.1 and 150 GPa, and asubstantially gas-permeable inner channel wall. In one embodiment, saidsubstantially gas-permeable inner channel wall comprisespolydimethylsiloxane (PDMS).

Despite these advantages and the ability to modify experimentalprotocols to enable these low-absorbing, gas-impermeable microfluidicdevices, they do suffer from a lack of ambient gas flow. Some commonlyused microfluidic devices are fabricated out of elastomeric orgas-permeable materials, such as PDMS and other gas-permeable or porousor elastomeric materials. Scientists oftentimes rely on thegas-permeable characteristic of these materials, such as PDMS, in orderto introduce ambient gases through the bulk material of the microfluidicdevice. In some cases, entirely gas-impermeable microfluidic devices maycause harm to specimens, such as cells, as they are unable to accessambient gases, such as oxygen. Certain specimen, such as some varietiesof cell cultures, require specific levels of particular gases in orderto be physiologically relevant or even in order to survive. However,experimental protocols may be adapted such that higher levels of desiredgas may be introduced into the low-absorbing, gas-impermeablemicrofluidic devices, through the use of techniques such as higher flowrates and the use of medias with higher gas concentrations.

There are several methods contemplated to increase gas delivery intomicrofluidic devices. These methods include increasing the flow rate ofmedia into the microfluidic device to increase the volume ofgas-containing media flowing over the cells with time, increasing thedissolved gas content of the media flowing through the microfluidicdevice, recirculation of media, reciprocation of media from environmentswith high concentrations of ambient gases to thegas-impermeable/gas-consuming interior of the device and then backagain, and delivering gases to the interior of the microfluidic devicefrom the outside ambient environment through the microfluidic devicebulk material.

As such, the present invention contemplates several protocols to usethese rigid or low-absorbing, gas-impermeable microfluidic devices. Afirst protocol is to perfuse these rigid or low-absorbing,gas-impermeable microfluidic devices with high flow rates of fluid ormedia in order to transport gases into the channels of the microfluidicdevice via the dissolved gasses of the fluid or media. In oneembodiment, increased gas transport into the microfluidic device may beachieved by using higher flow rates of media containing the importantgases, such as oxygen, into the microfluidic device. In this embodiment,as the flow rate of the media is higher, more media is introduced intothe microfluidic device in a set amount of time, and thus more of thedesired gas is introduced into the microfluidic device. The use of highflow rates in microfluidic devices to increase gas transport is usefulin gas-impermeable microfluidic devices, as gas may not intrinsicallydiffuse into the microfluidic device otherwise. Increasing flowrate alsoremoves unwanted gasses, such as carbon dioxide produced by the cells asa byproduct of respiration. Interestingly, increased flow rates may alsosignificantly improve compound distribution within a microfluidicdevice, such that the majority of the small molecules are not absorbedin the first portion of a channel. Those conducting these experimentswere surprised by how effective increasing the flow rate, even slightly,was at improving the distribution of the agent, drug, etc. throughoutthe channel.

Oftentimes these rigid or low-absorbing, gas-impermeable microfluidicdevices need to be perfused with media nearly continuously in order todeliver the oxygen required for particular experiments, such as thosecomprising cells undergoing cellular respiration; the oxygen isdelivered via the oxygen dissolved in the media entering themicrofluidic device as opposed to what may be considered the moreefficient route of a gas-permeable microfluidic device, which isdirectly through the bulk material. The efficiency is forsaken in orderto limit molecule absorption into the body of the microfluidic device.For example, if microfluidic devices comprising cells remain static,that is without perfusion with oxygen carrying media, for an extendedperiod of time, cells contained within loose viability and function, insevere cases apoptosing or necrosing. In a surprising finding, it wasdiscovered that in the case of liver hepatocyte cells cultured withinmicrofluidic devices, the hepatocytes experience the negativeconsequence of oxygen deprivation within minutes of stopping fluid flow.Hepatocytes are a highly metabolically active cell type. Similarly, itwas found that to deliver enough oxygen to ensure cell viability andfunction in low-absorbing, gas-impermeable microfluidic devices, that itwas necessary to perfuse the devices at flow rates that were higher thantypically used for the culture of hepatocytes. To mitigate thischallenge, higher flow rates than are typically used for the culture ofhepatocytes in these microfluidic devices have been utilized and thetime that the low-absorbing, gas-impermeable microfluidic devicesremained static, that is without media perfusion, was minimized. It wasdiscovered through experimentation that many of the cell seeding stepsthat typically are performed under no fluid flow or static conditionscould be shortened and that higher flow rates than were typically usedcould be utilized in the devices. For example, it was found that theserigid or low-absorbing, gas-impermeable microfluidic devices do not needto remain static for extended periods of time for adequate cellattachment to the microfluidic device. For example, for certainexperiments microfluidic devices may need to be inverted without flow inorder to allow cells within the microfluidic device to attach to amembrane. In this case, the cells would need to be without flow, andtherefore without oxygen they require. However, even for this case itwas found that the microfluidic devices do not necessarily need to beinverted without flow for extended periods of time. Surprisingly, evenconnecting these low-absorbing, gas-impermeable microfluidic devices toflow, even high flow, immediately following seeding had minimal negativeconsequences on the cells cultured within the device. Occasionalhigh-flow cycles, which may be used to dispose of bubbles, may also beused on the low-absorbing, gas-impermeable microfluidic devices with noapparent negative consequences to cell layers within. Additionally, itwas found that several time-intensive and sequential steps could becombined to save time and minimize the time that microfluidic devicesremained static, including seeding endothelial cells and applyingextracellular matrix in the same step, condensing the overall seedingprotocol, making the use of these microfluidic devices moreuser-friendly and advantageous over other microfluidic device designs aswell.

In one embodiment, a method of controlling gas transport iscontemplated, comprising: a) providing a substantially gas-impermeablemicrofluidic device comprising a rigid polymer and living cells in achannel or chamber, said living cells having a gas consumption rate; andb) introducing culture media into said channel or chamber at a flowrate, said culture media carrying gas, wherein the rate of gas transportto said living cells is controlled by the flow rate, and said rate ofgas transport meets or exceeds said gas consumption rate. In oneembodiment, said rigid polymer has a modulus of elasticity between 0.1and 150 GPa. In one embodiment, said rigid polymer is polycarbonate. Inone embodiment, said flow rate is greater than 40 uL/hr. In oneembodiment, a method further comprises increasing the flow rate in orderto increase the rate of gas transport. In one embodiment, a methodfurther comprises introducing a drug or drug candidate into said channelor chamber, wherein said rigid polymer reduces the absorption of saiddrug or drug candidate by at least about 70% or more, as compared to theextent of absorption into PDMS. In one embodiment, a method furthercomprises evaluating the viability of said cells via cellular assaysand/or visual inspection.

However, increasing the flow rate of media into the microfluidic devicemay not be physiologically relevant, as fluids in vivo flow at specificflow rates. It is usually desired to expose specimen, such as cells, tosimilar conditions in vitro as is found in vivo. Fluid flow rate isdirectly related to shear; increasing the flow rate of media into themicrofluidic device may expose specimen, such as cells, to undue levelsof shear. Undue levels of shear may have negative impacts, particularlywhen the high shear is in channels containing cells that typically areexposed to low or no shear/fluid flow. The flow rate of media throughthe device impacts the ability of cells to communicate with one othervia cellular factors secreted into the microfluidic channel. Inparticular, at excessively high flow rates, these cytokines and otherdissolved factors are diluted into the high volume of media passingthrough the microfluidic device. High flow rates effectively wash outthe factors and can prevent the cells form sensing the signals and,therefore, hinder or prevent the proper in vivo response that isattempting to be recapitulated. Similarly, just as signaling factors canbe diluted out by the higher flow rates, so too can the various factorsthat are excreted by cells into the effluent media that are beingquantified. For example, if rate of metabolism is being assessed, theconcentration of the metabolized form of the dosed compound might be sodilute that it is effectively undetectable in the media as theconcentration falls below the lower limit of detection of the analyticalinstrument. If metabolism is being detected by depletion of the compoundbeing dosed, a high flow rate will decrease the change in concentrationof the parent compound as it passes through the microfluidic device,which also can make detection of this change impossible. In other words,high flow rates can decrease the “signal” that is being sensed to thelevel of the noise of the analytical instrument, making quantificationimpossible. The principle is similar for cell-to-cell signaling; thereleased factors are diluted below the concentration that other cellscan detect and respond to. Finally, since cells within thelow-absorbing, gas-impermeable microfluidic device are supplied withoxygen via media flow a major limitation of this approach is that thecells within the device do not receive oxygen if the low-absorbing,gas-impermeable microfluidic device is removed from fluid flow. Notbeing able to remove the low-absorbing, gas-impermeable microfluidicdevice from flow without negative consequences on the cells withinpresents a major practical limitation, since microfluidic devices oftenrequire periodic periods where flow is stopped such that the cellswithin the device can be imaged under a microscope and also to refillinlet and outlet media reservoirs of perfusion manifold assemblies withmedia. Indeed, delivery of oxygen to the cell layer is vital to thebasic function of the cells and necessary to maintain viability; ifmedia flow is stopped for an extended period of time, the cells withinthe rigid, low-absorbing, gas-impermeable microfluidic device tend todie.

Considering the disadvantages of running fluid at high flow rates inmicrofluidic devices seeded with cells other alternatives weredeveloped. Another embodiment of the application herein is a protocolfor using the rigid microfluidic devices of U.S. patent application Ser.No. 15/105,388 with recirculated fluid. In this embodiment, fluid thatexits the microfluidic device may be recirculated to the inlet of themicrofluidic device. Not only does this setup decrease the amount offluid or media necessary, but also preserves any important chemical orbiochemical markers within the media that would be beneficial ifreintroduced into the microfluidic device. For example, cells secretesignals, such as paracrine and autocrine signals. It is advantageous tohave cell signals not wasted, but instead continuously in contact withthe cells.

In one embodiment, one or more microfluidic devices are contemplatedcomprising i) cells on a surface and ii) inlet and outlet ports, saidinlet and outlet ports in fluidic communication with a recirculationpathway, and 2) flowing culture media into said one or more microfluidicdevices in a direction, thereby causing fluid to exit said outlet portof said one or more microfluidic devices and enter said recirculationpathway, moving in the direction of said inlet port of said one or moremicrofluidic devices. In one embodiment, said fluid moves in thedirection of said inlet port to reach said inlet port, therebyrecirculating said culture media without reversing the direction offluid flow. Secreted factors and waste products in recirculatingcultures are recirculated back to the cells, whereas innon-recirculating culture, the secreted factors and waste products arepermanently removed. Where recirculation is desired, a given volume ofculture media (e.g. all of it, a portion of it, etc.) is recirculated,whereas in non-recirculating perfusion, the culture media is perfusedthrough the system and sent to directly to waste. Secreted factors andwaste products in recirculating cultures are diluted into the totalculture media volume (although this can be avoided by the use of asecond reservoir, and the second reservoir can be avoided by usingtubing). In one embodiment, media flowed through the low-absorbing,gas-impermeable microfluidic device may be collected and recirculatedback through the low-absorbing, gas-impermeable microfluidic device. Inone embodiment, the media is recirculated once. In another embodiment,the media is recirculated more than one. Recirculating media solvesthree problems: specimen within microfluidic devices may be furtherexposed to experimental compounds dosed in the media, the media may berefilled with nutrients in between recirculation, and both cellsignaling factors and factors to be analytically quantified as cellularreadouts will not be diluted out by the high-volumes required for thesingle-pass, high flow rate solution. Increasing the flow rate does notsolve the practical problem that once the device is removed form fluidflow, the delivery of oxygen and removal of CO₂ ceases.

In one embodiment of a recirculation setup or method, it is contemplatedthat the media would flow through low-absorbing, gas-permeable tubingwhere it could come into contact with ambient gases before flowing intothe microfluidic device. The media, having been depleted of thoseambient gases while inside the microfluidic device by the specimen,would then rapidly equilibrate to the ambient environment.

In one embodiment, when the media is flowed past cells, the oxygenconcentration in the media is depleted due to cellular respiration. Inone embodiment of maintaining both oxygen levels in the desired flowrate within a microfluidic device, a recirculation experimental setupmay be used. In one embodiment, a recirculation setup is used such thatthe media can be re-oxygenated prior to being recirculated through themicrofluidic device and CO₂ removed from the media. In anotherembodiment, a recirculation setup is used such that cells within thelow-absorbing, gas-impermeable microfluidic device experience prolongedexposure to a dosing compound while maintaining a desired flow rate. Inthis setup, the low volume enabled by reciprocation enables the longexposure time. In one embodiment, cells requiring a high shear rate arebeing dosed with a low clearance compound. Low clearance compounds aremetabolized slowly be cells. High flow rates may be used to produce highshear force on cells and to increase the amount of oxygen delivered tothe cells. However, if a high flow rate is used, the cells are generallynot exposed to low clearance compounds long enough for a significantamount of metabolism to take place and quantification of thismetabolism, let alone detection, is impossible. The recirculation setupmay be used to maintain high flow rates, while still allowing cells tobe exposed the low clearance compound long enough to detect and quantifymetabolism.

In another embodiment, the dissolved gas content of the media flowingthrough the microfluidic device may be increased prior to it enteringthe microfluidic device. In one embodiment, the dissolved gas content ofthe media may be increased prior to entering the microfluidic device bybubbling gas through the media. The content of this gas mixture can bedetermined based on the aim attempting to be achieved; if the aim is toincrease dissolved oxygen, then a gas mixture containing a highconcentration of oxygen can be utilized (e.g. bubbling 100% oxygenthrough the media increases the media oxygen content by a factor of 5compared to atmospheric air which is only 21% oxygen). In anotherembodiment, the dissolved gas content of the media may be increasedprior to entering the microfluidic device by pressurizing the media withthe desired gas or a carrier of the desired gas. Atmospheric pressure is˜101 kPa—by pressurizing with 202 kPa, as an example, the gasconcentration is increased by a factor of two. In one embodiment theconcentration of oxygen within a media may be increased by pressurizingthe media with a higher oxygen concentration than atmospheric levels orby using an oxygen carrier within the media, such as hemoglobin,perfluorocarbon-based oxygen carriers, hemocyanin, etc. In oneembodiment, the gas pressurizing the media may be a gas blanket.However, simply increasing the dissolved gas content of the media maynot be physiologically relevant as fluids in vivo contain specificconcentrations of gas. It is usually desired to expose specimen, such ascells, to similar conditions in vitro as is found in vivo. Oxygencarriers generally do not suffer from this limitation, since theyincrease the oxygen carrying capacity without increasing the dissolvedoxygen (the additional oxygen is bound to the carrier and not dissolvedin the media). Both flowing media at higher flow rates and increasingthe dissolved gas content of media also succumb to the followingsignificant shortfall. As the media flows through the microfluidicdevice, the specimen at the beginning of the channels will experiencehigher levels of the desired gas. The specimen at the beginning of thechannel may then uptake high levels of said gas, leaving lower levels ofthe desired gas for specimen further in the channels. The spatialgradient in cellular oxygen exposure levels can result in gradients incellular response, which are difficult to assess since the effluent fromthe microfluidic devices is a pooled sample of the media as it passesthrough the microfluidic device. This solution, too, does not solve thepractical problem that once perfusion of media stops, so too does thedelivery of oxygen and removal of CO₂.

However, recirculation systems can be difficult to setup and use. Forexample, peristaltic pumps are oftentimes necessary for recirculationsetups. Peristaltic pumps disadvantages include size limitations andthat they oftentimes require flexible tubing. Flexible tubing isoftentimes made of elastomeric polymers. As stated before, elastomericpolymers are prone to material absorption. As such, one embodiment ofthe present invention is a protocol for using the rigid microfluidicdevices of U.S. patent application Ser. No. 15/105,388 with reciprocatedfluid. For uses herein, reciprocation is flowing a fluid in onedirection through a microfluidic device, collecting that fluid, and thenflowing the same fluid in the other direction through the microfluidicdevice. Reciprocation of fluid or media through a microfluidic device isnot obvious or intuitive fluids are not reciprocated through the body.However, surprisingly cells in microfluidic devices experienced highlevels of viability when media was reciprocated in the microfluidicdevices. When fluid is reciprocated in microfluidic devices, simpletwo-way pumps may be used, low volumes of media may be used, ambient gaslevels may be introduced to the channels of the microfluidic device, andthe media retains signals secreted by the cells.

The present invention contemplates, in one embodiment, a method ofcontrolling gas transport, comprising: a) providing a substantiallygas-impermeable microfluidic device comprising i) living cells on asurface and ii) inlet and outlet ports, said inlet and outlet ports influidic communication with iii) a recirculation pathway, and b) flowingculture media carrying gas at a flow rate into said inlet port of saidmicrofluidic device in a direction, thereby causing fluid to exit saidoutlet port of said microfluidic device and enter said recirculationpathway, thereby recirculating said culture media without reversing thedirection of fluid flow, wherein the rate of gas transport to saidliving cells is increased by said recirculating. In one embodiment, saidrigid polymer has a modulus of elasticity between 0.1 and 150 GPa. Inone embodiment, said flow rate is 40 uL/hr or less.

The present invention contemplates, in one embodiment, a method ofcontrolling gas transport, comprising: a) providing a substantiallygas-impermeable microfluidic device comprising i) living cells on asurface and ii) inlet and outlet ports, said inlet and outlet ports influidic communication with iii) a reciprocation actuator, and b) flowingculture media carrying gas at a flow rate into said inlet port of saidmicrofluidic device in a direction, thereby causing fluid to move in thedirection of said outlet port; and c) reciprocating said fluid with saidreciprocation actuator, thereby reversing the direction of fluid flow,wherein the rate of gas transport to said living cells is increased bysaid reciprocating. In one embodiment, said rigid polymer has a modulusof elasticity between 0.1 and 150 GPa. In one embodiment, said flow rateis 40 uL/hr or less.

Despite the benefits of high flow rates, recirculation andreciprocation, occasionally these rigid microfluidic devices need to beremoved from flow in order to take samples, image under microscopes, addnew cell types, etc. It has been found that even after a few minuteswithout flow, some cell types within microfluidic devices begin tosuffer from a lack of oxygen. Following this discovery, a microfluidicdevice fabricated from a strategic combination of rigid and elastomericpolymers was developed in order to ensure that both the microfluidicdevice is able to stretch, but almost more importantly that the channelswithin the microfluidic device are able to access ambient gases.Reciprocation is taught in International Patent Application No.PCT/US2019/25449, the contents of which are incorporated herein byreference

If flow rate flexibility, and controlled, consistent,physiologically-relevant dissolved concentrations of gasses in media,and physically relevant environments are desired, and/or user-friendlyworkflows that allow for the microdevice to be removed from fluid flowfor short periods, then microfluidic devices that are both low-absorbingand gas-permeable would be advantageous. As such, microfluidic devicesfabricated that are both low-absorbing, but have controllablegas-permeability would be advantageous compared to completelygas-impermeable microfluidic devices as they as they decrease absorbencyof important compounds being tested as well as allow the cells to accessambient gases during the experiment. In one embodiment a microfluidicdevice fabricated out of a strategic combination of gas-permeable andgas-impermeable materials is contemplated. A low-absorbing,gas-permeable microfluidic device was then desired based on the resultsgathered using the low-absorbing, gas-impermeable microfluidic device.In some embodiments, the material makeup of this microfluidic device canreduce absorption of molecules by at least about 10% or more, including,e.g., at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90% or more, as compared to the extent of moleculeabsorption into PDMS.

A resulting low-absorbing, gas-permeable microfluidic device iscontemplated in one embodiment, comprising a body having at least onechannel therein, said channel having channel walls and a membrane,wherein at least one of said channel walls and membrane are elastomeric.The microfluidic device may be predominantly rigid, while having achannel comprising elastomeric walls and an elastomeric membrane. Themembrane may be elastomeric to facilitate gas transport on either sideof said membrane. The walls of the channel may be elastomeric tofacilitate stretching of the membrane if desired. However, in someembodiments differential pressure may be used to stretch said membrane,and in that case the body and channel walls may be rigid, while simplythe membrane is elastomeric. In the embodiment where solely the membraneis elastomeric, the amount of absorbing material may be minimized as themembrane may represent a small volume of the membrane in one embodiment.In one embodiment, the microfluidic device comprises a body having atleast one channel therein, said channel having elastomeric walls and anelastomeric membrane, wherein at least a portion of said body is rigid.Furthermore, the embodiment comprising elastomeric channel walls and amembrane may necessitate further fabrication steps than an embodimentwherein the body is entirely rigid. In one embodiment, the microfluidicdevice comprises a body having at least one channel therein, saidchannel having rigid walls and an elastomeric membrane, wherein at leasta portion of said body is rigid.

The present invention contemplates, in one embodiment, another resultinglow-absorbing, gas-permeable microfluidic device such as a microfluidicdevice comprising a body, said body having a channel therein, and a gasexchanger. A low-absorbing microfluidic device may comprise a rigid bodyand a gas exchanger, such that the body does not absorb molecules, whilegas transport can still take place within the microfluidic device. Whilethe gas exchanger may be in any portion of the microfluidic device, inan exemplary embodiment the gas exchanger is in contact with a channel,such that gas may be exchanged from the ambient environment with a cellculture in the channel.

In one embodiment, the low-absorbing, gas-permeable microfluidic devicecomprises a low-absorbing body having a channel, said channel having achannel wall, wherein said channel wall comprises a gas exchanger incontact with the ambient environment. In one embodiment, thelow-absorbing, gas-permeable microfluidic device comprises alow-absorbing, rigid body having a channel, said channel having achannel wall, wherein said channel wall comprises a gas exchanger havinga gas-permeable material in contact with the ambient environment.

In one embodiment, the low-absorbing, gas-permeable microfluidic devicecomprises a solid substrate comprising one or more microfluidicchannels. In one embodiment, the microfluidic device comprises aplurality of microfluidic channels. In an exemplary embodiment, thelow-absorbing, gas-permeable microfluidic device comprises: a) a solidsubstrate comprising a single microfluidic channel, b) a porous membraneseparating said single microfluidic channel into a first chamber and asecond chamber, and c) a gas exchanger to allow gas transport from theambient environment outside the microfluidic device into themicrofluidic device. It is not intended that the microfluidic device belimited by substrate, membrane, chamber or channel configuration. In oneembodiment, said first and second chambers are oriented vertically. Inone embodiment, said first and second chambers are orientedhorizontally. Said first and second chambers may also be referred to aschannels. Said first and second chambers, if oriented horizontally, maybe referred to as top and bottom chambers or channels.

In one embodiment, the microfluidic device is fabricated from a firstand second channel layers. Said first channel layer may comprise a firstsurface and a second surface. Said second channel layer may comprise athird surface and a fourth surface. Microfluidic chambers or channelsmay be disposed upon said surfaces. For example, chambers may be etched,molded, or cut onto substrate surfaces. In one embodiment, said firstsurface comprises said first chamber. In one embodiment, said thirdsurface comprises said second chamber. Said first channel layer may bereferred to as a first layer or first substrate. Said second substratemay be referred to as a second layer or second channel layer. If thefirst and second chambers or channels are oriented vertically, saidfirst channel layer may be referred to as a top layer or top substrate.If the first and second chambers or channels are oriented vertically,said second channel layer may be referred to as a bottom layer or bottomsubstrate.

In an exemplary embodiment, the membrane may be sandwiched between thefirst and second channel layers. The first and second channel layers,the membrane and the gas exchanger may be attached permanently ortemporarily. A first fluid may be applied through said first chamber. Asecond fluid may be applied through said second chamber. In oneembodiment the layers are attached through plasma-activated bonding.Unlike the microfluidic device presented in U.S. Pat. No. 8,647,861, themicrofluidic device presented here may only optionally contain workingchannels for mechanical actuation.

In one embodiment, the microfluidic device is used for thecharacterization of organ microbiomes. In one embodiment, thelow-absorbing, gas-permeable microfluidic device may be used to test theeffects drugs, foods, chemicals, cosmetics, physiological stimulantsstresses etc. have on cellular systems. Different cell types sometimesrequire different amounts of oxygen in order to thrive. If cellularhealth is a goal, oxygen entering the device should be greater thanoxygen uptake rate within the microfluidic device in order to ensurethat cells have access to as much oxygen as they require. For example,liver hepatocytes oftentimes require atmospheric levels of oxygen,whereas some bacteria cultures in the gut require very little oxygen. Assuch, microfluidic devices, especially those with applications incellular biology, would benefit by being low-absorbing, while stillallowing necessary levels of oxygen to reach cells, experiments, etc.inside the microfluidic device. Oftentimes however, low-absorbingmaterials tend to be gas-impermeable. In this way, a microfluidic deviceminimizing the amount of material absorbency may be designed with acombination of gas-permeable and gas-impermeable components.

Another important aspect of microfluidic device material choice to beconsidered is transparency. Optical transparency is advantageous inmicrofluidic devices for multiple reasons. Transparency is advantageousfor imaging. In one embodiment, the low-absorbing, gas-permeablemicrofluidic device described here may be used in conjunction with amicroscope, such as an inverted microscope, an upright microscope, aconfocal microscope, a light microscope, an electron-scanningmicroscope, etc. Transparency is also advantageous with the use ofoptogenetically active cells. In one embodiment, the cell layer in thelow-absorbing, gas-permeable microfluidic device comprises a layer ofoptogenetically active cells. In one embodiment, the materials making upthe microfluidic devices are also biocompatible. As an exemplary use ofthe microfluidic devices presented herein is for the use of culturingcells, biocompatibility may be important.

Imaging microfluidic devices on microscopes enables scientists to get anintimate perspective on cellular interactions, phenotypes, and more.Opaqueness offers scientists the ability to protect their experimentsfrom ambient light if necessary. In one embodiment, the low-absorbing,gas-permeable microfluidic device is fabricated from opaque materials.As such, the microfluidic device presented herein may be partially orentirely transparent or entirely opaque depending on the needs of theexperiment and the particular embodiment.

First and second channels layers comprise substrates containing one ormore channels or pathways for fluid movement and experiment housing.Each channel layer may comprise one or more microfluidic channels. Inanother embodiment, each channel layer may comprise a plurality ofchannels. In one embodiment, where the microfluidic device is assembledin a vertical orientation, the first channel layer may be considered thetop channel layer and comprises a first or top channel and the secondchannel layer may be considered the bottom channel layer and comprises asecond or bottom channel. In particular embodiments, the first channelmay be referred to as the top channel due to its location above amembrane and the second channel may be referred to as the bottom channeldue to its location below a membrane. In such an embodiment, a membraneseparates the first and second channels.

Experiments contained within the channels include cell growth andtesting. In one embodiment, cells are grown in the channels of thelow-absorbing, gas-permeable microfluidic device as to form a celllayer. In one embodiment, epithelial cells are grown in the firstchannel in the top channel layer and endothelial cells are grown in thesecond channel in the bottom channel layer. In one embodiment,epithelial and endothelial cells cultured within said first and secondchannels are separated by a membrane.

Channels in the channel layers may be a variety of different heights,including but not limited to equaling the height of the channel layeritself or cutting through the entire channel layer. In one embodiment,the height of the first channel is less than the height of the channellayer comprising the channel. In one embodiment, the height of thesecond channel is less than the height of the channel layer comprisingthe channel. The heights of a first channel and a second channel canvary to suit the needs of desired applications. In one embodiment, thechannel heights are chosen to suit a particular cell size. In oneembodiment, the channel heights are chosen to suit a particular shearforce level. In one embodiment, channel heights are between 10 μm and5000 μm. In one embodiment, the channel heights are between 100 μm and1000 μm. In one embodiment, a “regular” channel height may be 100 μm,while a “tall” channel height may be 1000 μm. In one embodiment, theheight of a first channel is equal to the height of the channel layercomprising the channel. In one embodiment, the height of the secondchannel is equal to the height of the channel layer comprising thechannel. In one embodiment, the height of a first channel and the heightof a second channel are the same. In one embodiment, the height of afirst channel and the height of a second channel are different. In oneembodiment, the height of a first channel is greater than the height ofa second channel. In one embodiment, the height of the first channel is1000 μm and the height of the second channel is 100 μm. In oneembodiment, the height of a first channel is greater than the height ofa second channel because epithelial cells are seeded in the firstchannel, while endothelial cells are seeded in the second channel. Somevarieties of epithelial cells are larger, and therefore may require moreroom than endothelial cells. Particular ratios of the first channel andsecond channel heights are advantageous for particular cell lines andlevels of shear force. In one embodiment, the height of a first channelis consistent across the entire first channel. In one embodiment, theheight of a second channel is consistent across the entire secondchannel. In one embodiment, the height of a first channel isinconsistent along the length of the first channel. In one embodiment,the height of a second channel is inconsistent along the length of thesecond channel. In one embodiment, the height of a first channel islarger in the cell culture area, as compared to non-cell culture areaswithin the first channel. In one embodiment, the height of a secondchannel is larger in the cell culture area, as compared to non-cellculture areas of the second channel. For example, the height ratio ofthe first channel to the second channel is greater than 1:1, including,for example, greater than 1.1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1,4.5:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1,16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1. Insome embodiments, the height ratio of a first channel to a secondchannel can range from 1.1:1 to about 50:1, or from about 2.5:1 to about50:1, or from 2.5 to about 25:1, or from about 5:1 to about 25:1. In oneembodiment, the height ratio of a first channel to a second channelranges from about 10:1 to about 20:1.

Different embodiments comprise microfluidic devices with differentchannel alignments. Channels may be aligned differently to achievevarious levels of cellular interaction. For example, if cells arecultured in channels one two opposing sides of a membrane, the channelson either side of the membrane may be aligned such that they onlyoverlap 50%, such that only 50% of the cells may interact with eachother. In one embodiment, a first channel on a first side of a membraneand a second channel on a second side of a membrane are aligned. In oneembodiment, the first and second channels, on opposing sides of amembrane, are not aligned. In one embodiment, the first and secondchannels are partially aligned. In one embodiment, there is a port orvia at both ends of a first or second channel so that fluids may beintroduced into the microfluidic device. In one embodiment, microfluidicdevice infrastructure may be made to be in fluidic communication withthe microfluidic device through these ports. First and second channellayers may be fabricated from the same or different materials. In oneembodiment, the first, or top, channel layer and second, or bottom,channel layers are fabricated from the same material. In one embodiment,the first channel layer and second channel layer are fabricated fromdifferent materials. In one embodiment, the first channel layer is madeup of a single material. In one embodiment, the second channel layer ismade up of a single material. In one embodiment, the first channel layeris made up of multiple materials. In one embodiment, the second channellayer is made up of multiple materials. In one embodiment the firstchannel layer is fabricated from one or more gas-permeable materials. Inone embodiment, the first channel layer is fabricated from one or moregas-impermeable materials. In one embodiment, the second channel layeris fabricated from one or more gas-permeable materials. In oneembodiment, the second channel layer is fabricated from one or moregas-impermeable materials. Gas-impermeable materials that have also beenshown to be low absorbing include cyclic olefin copolymer (CCP), cyclicolefin polymer (COP), polycarbonate, polyethylene (PE), polyethyleneTerephthalate, polystyrene (PS), (PET) glass, etc. In one embodiment,the first and second channel layers are fabricated fully or partiallyfrom gas-permeable materials and are modified in such as a way as tolimit absorbency. In one embodiment, the first and second channel layersmay achieve low-absorbency by being fabricated partially fromgas-impermeable materials. In one embodiment, the first and secondchannel layers may achieve low-absorbency by being coated with asubstance. In one embodiment, the first and second channel layers mayachieve low-absorbency by having their surfaces modified to reachimpermeability.

The membrane provides a diffusive barrier between first and secondchannels on opposing sides of the membrane. While the membrane may begas-impermeable, oftentimes it is beneficial to allow oxygen diffusionthrough the membrane. As such, in one embodiment, the membrane isgas-permeable. In one embodiment, the membrane is fabricated from PDMS.However, one embodiment, the membrane is gas-impermeable. For example,cell types in the top and bottom channel may benefit from exchanginggases. Gas-permeability may be prioritized over low-absorbency in themembrane layer for this diffusivity reason. In some embodiments, themembrane may be a smaller volume as compared to the volumes of othercomponents of the microfluidic device, such as the top and bottomchannel layers and the gas exchanger. If the membrane has a smallervolume than other components it would not absorb as much of theexperimental compound, minimizing absorbency impacts. In otherembodiments, the membrane is non-porous in order to limit physicalcontact between top and bottom channel environments and inhabitants. Insome embodiments, the membrane is porous in order to allow contactbetween top and bottom channel environments and inhabitants. In oneembodiment, the membrane layer is homogenous, such as beingevenly/porous across the entire layer. In another embodiment, themembrane layer is heterogenous, such as being porous only in the regionsthat overlap top and bottom channels. In some embodiments, the membraneis flexible as to allow it to stretch. In this embodiment, the abilityto stretch, or achieve actuation, is beneficial for experimentsinvolving cells attached to the membrane, as it is able to replicatemechanical strain on in-vivo cells. In some embodiments, stretch, oractuation, is achieved by using vacuum in optional working channels inthe microfluidic device. In some embodiments stretch, or actuation, isachieved by having a pressure differential in the top and bottomchannels, as to push the membrane in the direction of the lower pressurechannel. Stretch or actuation achieved by a pressure differential may beadvantageous as it may be more physiology relevant than actuation of themembrane by vacuum channels which applies no pressure to the cell layer.Indeed, this stretching mechanism better recapitulates the physiologicmechanisms for mechanical stretching of cells and tissues, which includepressure differentials. For example, arteries tend to expand as theheart beats and expels blood from within the ventricles and into theartery lumen. This expansion (and resulting strain on the cellscomposing the vasculature walls) occurs because of the pressuregenerated by the beating heart, much like a balloon expands whenpressurized with air. The pressures needed to flex the membrane andcreate these in vivo relevant strains is, in one embodiment, a similarpressure as would be seen in the capillary beds of the lungs. Statedmore simply, in one embodiment both the pressures that the cell layersare exposed to and the stretch are tuned to be simultaneouslyphysiologically relevant. Additionally, the shape of this stretch betteremulates the shape of the expansion seen in blood vessels and thealveolar sacs, since in this embodiment the membrane is physicallydisplaced into a channel and assumes the shape of an arc as opposed to alinear displacement (i.e. the membrane moves up and down as itstretches).

As previously stated, in order to overcome low levels of important gasesin microfluidic devices, as well as avoid the use of high, continuouslyapplied, flow rates and high dissolved gas concentrations in the media,a gas exchanger may be built into the microfluidic device in such a wayas to not promote molecule, substance and/or experimental compoundabsorbency while still allowing important gases, such as oxygen, todiffuse through the microfluidic device. In one embodiment, thelow-absorbing, gas-permeable microfluidic device comprises agas-exchanger. In one embodiment, the purpose of the gas-exchanger is tointroduce ambient gases into the microfluidic device. In one embodiment,the purpose of the gas-exchanger is to introduce selected gases into themicrofluidic device.

In one embodiment, a gas exchanger may be incorporated into themicrofluidic device as a structural element of the microfluidic devicein contact with the ambient environment or a desired gas source. In oneembodiment, the gas exchanger may be channel capping layer, such thatthe gas exchanger encloses one or more channels within the microfluidicdevice. As such, in one embodiment, the gas exchanger caps one or morechannels. In one embodiment, the gas exchanger is attached to the bottomof the microfluidic device, such as to form a floor to the bottomchannel layer. In this embodiment, the ceiling of the bottom channelwould be the membrane and the base of the bottom channel would be thegas exchanger.

In one embodiment, the gas exchanger may comprise a single material or acombination of materials. Materials used to fabricate the gas exchangermay be selected from polydimethylsiloxane (PDMS), room temperaturevulcanizing (RTV) silicone, TeflonAF2400, polymethylpentene (PMP),polyethylene terephthalate (PET), polycarbonate (PC), cyclic olefinpolymer (COP), etc.

In one embodiment, room temperature vulcanizing (RTV) silicone may beused for the gas exchanger. In one embodiment, RTV silicone may besprayed onto the body of the microfluidic device to fabricate a gasexchanger.

In another embodiment, TeflonAF2400 may be used as a gas exchangermaterial. TeflonAF2400 is an exceptional material, as it is transparent,gas-permeable and low-absorbing to non-absorbing. In one embodiment, thegas exchanger may be fabricated out of a gas-permeable and/orgas-impermeable material and then coated with TeflonAF2400.

In another embodiment, polymethylpentene (PMP), commonly called TPX, atrademarked name of Mitsui Chemicals, may be used as a gas exchangermaterial. PMP or TPX is another exceptional material, as it istransparent, gas-permeable and low-absorbing.

Polymethylpentene (PMP) has several other advantageous properties, suchas favorable optical properties, a low cost, injection moldable, andresistant to many solvents. Resistance to solvents may be important ifthe microfluidic device is to be used during assays, as assays often useharsh solvents. A resistance to solvents may allow the microfluidicdevice to be used in a greater range of assays. Both TeflonAF2400 andPMP have the added advantage of being rigid materials and arestable/robust to the manual handling typically associated withmicrodevices. PMP may be fabricated in both liquid and solid form.

In some embodiments the inventors found that TPX or PMP can provedifficult to bond. As such, in other embodiments, the gas exchangercomprises PDMS. PDMS is advantageous as it is simple to use infabrication and bonds well. In one embodiment the gas exchanger may be alayer of PDMS. The PDMS may be applied using a variety of methods. Inone embodiment, a sheet or layer of PDMS may be applied to the body of amicrofluidic device. In one embodiment, the sheet or layer of PDMS maybe spin-coated. In one embodiment, the sheet of PDMS may be 2 μm±0.4 μm.In one embodiment, the PDMS may be coated onto said microfluidic device.In one embodiment, the PDMS may be spray coated on. With regard to thismethod, the inventors dissolved PDMS in a solvent and spray coated thebody of the microfluidic device. In one embodiment the gas exchanger isa thin layer of PDMS, such as to minimize molecule absorbance. However,the inventors found a thin layer of PDMS to be fragile in someinstances. In one embodiment, the gas exchanger is a thick layer ofPDMS, such as to be more durable. However, the inventors found a thickerlayer of PDMS to be more absorbent.

In one embodiment, the gas exchanger may be a combination of differentmaterials in order to overcome the above disadvantages. In an exemplaryembodiment of a gas exchanger, the gas exchanger may comprise acombination of a low-absorbing material with a gas-permeable material.The low-absorbing material may be porous, such that gas may flow fromthe ambient environment, through the gas-permeable material, and thenthrough the pores of the low-absorbing material.

In one embodiment, the gas exchanger is a two-layer combination of PDMSand polyethylene terephthalate (PET) or polycarbonate (PC). PDMS isgas-permeable and absorbent. PET is gas-impermeable and non-absorbent.In one embodiment, the PET may be porous.

In one embodiment, a gas exchanger may be fabricated and bonded to thebody of a microfluidic device in the exemplary protocol as follows. Asheet of PDMS may be spun coat to a thickness of 2 μm±0.4 μm. Theappropriate size may be cut out of the PDMS sheet. The sheet of PDMS maythen be bonded to a corresponding size of porous film, such as PET orPC. The bonding may be done via silane bonding. The compound(bis(3-triethoxysilypropyl)amine) may be mixed with 100% isopropylalcohol (IPA), which is then coated on the porous membrane before beinglet to dry. Once the mixture is dry, the PDMS may be plasma treated. Thetwo layers may then be adhered by contact.

In one embodiment one layer or material of the gas exchanger may beporous, as discussed above. In one embodiment, the porosity is createdthrough track etching. In one embodiment, the porosity of a component ofthe gas exchanger (such as PET or PC) is between 1% and 50%. In oneembodiment, the porosity is between 1% and 40%. In one embodiment, theporosity is between 1% and 5%. In one embodiment, the porosity is 1%. Inone embodiment, the porosity is 3%. In one embodiment, the porosity is11.4%. In one embodiment, the porosity is 40%. Porosities of 1%, 3%,11.4%, and 40% have all been explored with the microfluidic devicesherein. Porosities including but not limited to 0.3%, 1.6%, 3%, 5%,5.7%, 7.9%, 11%, 12.5%, 14.1%, 18.8%, 21.2% are commercially availableas well. Furthermore, porosities of any percentage may be fabricated andused. The porosity of the membrane may be tuned for the experiment. Theporosity of the membrane may be used to control the rate of oxygentransport within the microfluidic device. For example, based on theoxygen uptake rate of the cells within a microfluidic device, a membranewith a specific porosity may be used.

In the embodiment discussed above, track-etched PET serves as atransparent scaffold to give the gas exchanger mechanical stability andlow-absorbency, while the thin layer of gas-permeability PDMS seals thePET pores to leakage of fluid from inside the device to outside thedevice. The combination of PDMS and porous PET provides gas exchangingproperties, while having minimal absorption. In this embodiment, some ofthe small molecule compounds may absorb into the PDMS through the poresin the PET, however compared to the gas exchanger being fabricated froman entirely absorbent material, this absorbency may be considerednegligible in many cases. Further in this embodiment of the gasexchanger, the porous, track-etched PET and PDMS gas exchanger would notonly be able to increase gas transport compared to a completelygas-impermeable microfluidic device, but also decouples gas transportfrom fluid flow.

A gasket may be defined as a mechanical seal, which fills the spacebetween two mating surfaces, in order to, for example, prevent leaks orprovide compression. In one embodiment, the microfluidic device has agasket layer. In one embodiment, the gasket layer on the top surface ofthe microfluidic device. In one embodiment, the gasket layer has fourports to interact with the ports exiting a first channel of a firstchannel layer. In a particular embodiment, the gasket layer has fourports to interact with the ports exiting a top channel of a top channellayer. The gasket may be used to ensure a tight fluidic connectionbetween the microfluidic device and relating infrastructure. Theinclusion of a gasket layer is advantageous as it decreases the chanceof leakage compared to microfluidic devices not comprising a gasketlayer. In one embodiment the gasket is made out of a compressiblematerial. In another embodiment the gasket is made out of an adhesivematerial. The gasket may be used to keep the microfluidic device thesame size as it's absorbent predecessor in order to fit into existingmicrofluidic device accessories, such as a perfusion manifold. Thegasket may be embodied in multiple heights in order to raise the heightof the microfluidic device to a desired level such that it fits into acompression fit snugly. The gasket is not required to be gas-permeableand, therefore, may also be more easily made non-absorbent so that itdoes not absorb any small molecule compounds into the walls of itsports. The gasket may achieve non-absorbency by being fabricated from apartially or entirely gas-impermeable material, coated with agas-impermeable, non-absorbing substance, having its surface modified toreach impermeability (such as plasma treatment), etc. In one embodimentthe gasket covers the entire surface of the microfluidic device. Inanother embodiment the gasket only covers a portion of the surface ofthe microfluidic device.

In one embodiment the low-absorbing, gas-permeable microfluidic devicefeaturing a gas exchanger may be used to introduce and sustain a gasconcentration gradient in the microfluidic device. In this embodiment aspecific concentration of gas could be introduced to the gas exchanger.The gas is then depleted by the cell layers, such as endothelial andepithelial cell layers, resulting in a hypoxic first channel, topchannel, or luminal channel. In one exemplary embodiment the gas isoxygen. In another embodiment the gas is carbon dioxide. In anotherembodiment the gas is nitrogen. The gas gradient may be altered byintroducing cell layers of various permeability. The vertical gradientof gas through the microfluidic device maintains the longitudinalconcentration of gas along the entire length of the microfluidic device.In the embodiment where an oxygen gradient is introduced in thelow-absorbing, gas-permeable microfluidic device with a gas exchanger,the longitudinal oxygen concentration along the entire length of themicrofluidic device is maintained. In one embodiment, a gas-gradient isintroduced into the low-absorbing, gas-permeable microfluidic device byflowing the selected gas through adjacent working channels. In oneembodiment, a gas gradient is introduced into the low-absorbing,gas-permeable microfluidic device with a gas-exchanger using chemicalreactions. In another embodiment, the porosity of the PET scaffold isvaried to supply a greater flux of gas into and out of the microfluidicdevice.

In one embodiment, one or more sensors may be used to measure the gasgradient in the low-absorbing, gas-permeable microfluidic device. In theexemplary oxygen gradient embodiment, one or more oxygen sensors may beused to measure the oxygen gradient in the low-absorbing, gas-permeablemicrofluidic device. In one embodiment, the sensors are electricalsensors. In one embodiment the sensors are optical sensors. In oneembodiment, the one or more sensors are external to the microfluidicdevice. In one embodiment, the one or more sensors are inserted intoports or vias of the microfluidic device. In one embodiment, the one ormore sensors are embedded in the microfluidic device. In one embodiment,the one or more sensors are inserted into the material making up thebody of the microfluidic device. In one embodiment, the one or moresensors are in a first channel. In one embodiment, the one or moresensors are in a second channel. In one embodiment, the one or moresensors are in both a first channel and a second channel. In oneembodiment, the one or more sensors are in both a top channel and abottom channel. A plurality of sensors may be used in the microfluidicdevices presented herein, in order to measure gradients within saidmicrofluidic devices. A plurality of sensors may be used to measure anoxygen gradient. In one embodiment, sensors may be found along thelength of one or more channels within the microfluidic device, makingmeasurements, such as oxygen concentration measurements, along thelength of the channels.

The gas exchanger itself may be considered a unique invention. Gasexchangers have many uses, even outside the field of microfluidics. Gasexchangers may be helpful in transporting gases from one region another,or controlling the rate of gas exchange or flow. Gas exchangers may beused to maintain the rate of gas flow, decrease the rate of gas flow, orincrease the rate of gas flow.

A gas exchanger may be, in one embodiment, a gas-impermeable substratecomprising pores, wherein the pores are filled with a gas-permeablematerial. In an exemplary embodiment, the gas exchanger is fabricatedfrom a strategic combination of gas-impermeable and gas-permeablepolymers. The relative volumes of gas-permeable and gas-impermeablepolymers may be adapted in order to fabricate a gas exchanger ofdesirable characteristics. For example, if the gas exchanger is used toincrease the gas flow rate from, say, the ambient environment into amicrofluidic device, then a larger volume of gas-permeable polymer maybe used. For example, if the gas exchanger is used to decrease the gasflow rate from, say, the ambient environment into a microfluidic device,then a smaller volume of gas-permeable polymer may be used.

In one embodiment, a device is contemplated comprising a gas-impermeablesubstrate, said gas-impermeable substrate comprising (i) a firstsurface, (ii) a second surface, and (iii) one or more gas-permeableregions. In one embodiment, said substrate is a film. In one embodiment,said substrate is a sheet. In one embodiment, said substrate is alamination. In one embodiment, said substrate is a composite. In oneembodiment, said substrate is a gas-exchange membrane. In oneembodiment, said substrate is a gas-exchange membrane. In oneembodiment, said substrate is a pore-filled substrate. In oneembodiment, said substrate is a pore-filled film. In one embodiment,said substrate is a pore-filled gas-exchange membrane. In oneembodiment, said substrate is a pore-filled composite. In oneembodiment, said regions are pores. In one embodiment, said regions areconduits. In one embodiment, said regions are indentations. In oneembodiment, said regions contact at least one of said first surface andsaid second surface. In one embodiment, said regions bridge said firstsurface and said second surface. In one embodiment, said polymercomprises polyethylene terephthalate (PET) and said pores comprisespolydimethylsiloxane (PDMS).

In one embodiment, a rigid polymer film is contemplated comprisingelastomeric pores. In one embodiment, said rigid polymer film comprisespolyethylene terephthalate (PET) and said elastomeric pores comprisespolydimethylsiloxane (PDMS).

In one embodiment, the gas exchanger is a gas-impermeable substratecomprising one or more gas-permeable regions. In one embodiment, the gasexchanger is a rigid substrate comprising one or more flexible orelastomeric regions. In one embodiment, the gas exchanger is agas-impermeable substrate comprising a first surface, a second surface,and one or more gas-permeable regions. In one embodiment, the gasexchanger is a rigid substrate comprising a first surface, a secondsurface, and one or more elastomeric regions. In one embodiment, the gasexchanger is a gas-impermeable substrate comprising a first surface, asecond surface, and a one or more gas-permeable regions between saidfirst surface and said second surface. In one embodiment, the gasexchanger is a rigid substrate comprising a first surface, a secondsurface, and a one or more elastomeric regions between said firstsurface and said second surface. In one embodiment, the gas exchanger isa gas-impermeable substrate comprising a first surface, a secondsurface, and one or more gas-permeable regions contacting at least oneof said first surface and said second surface. In one embodiment, thegas exchanger is a rigid substrate comprising a first surface, a secondsurface, and one or more elastomeric regions contacting at least one ofsaid first surface and said second surface.

In one embodiment, said gas-exchanger is a gas-impermeable membranecomprising pores, wherein said pores are at least partially filled witha gas-permeable material. In one embodiment, said gas-exchanger is agas-impermeable film comprising pores, wherein said pores are at leastpartially filled with a gas-permeable material. In one embodiment, saidgas-exchanger is a rigid membrane comprising pores, wherein said poresare at least partially filled with an elastomeric material. In oneembodiment, said gas-exchanger is a gas-impermeable film comprisingpores, wherein said pores are at least partially filled with agas-permeable material. In one embodiment, said gas-exchanger is a rigidfilm comprising pores, wherein said pores are at least partially filledwith an elastomeric material. In one embodiment, said gas-exchanger is agas-impermeable polymer membrane comprising pores, wherein said poresare at least partially filled with a gas-permeable polymer. In oneembodiment, said gas-exchanger is a rigid polymer membrane comprisingpores, wherein said pores are at least partially filled with anelastomeric polymer. In one embodiment, said gas-exchanger is agas-impermeable polymer film comprising pores, wherein said pores are atleast partially filled with a gas-permeable polymer. In one embodiment,said gas-exchanger is a rigid polymer film comprising pores, whereinsaid pores are at least partially filled with an elastomeric polymer.

In one embodiment, the gas exchanger is a gas-impermeable substratecomprising a first surface, a second surface, and one or moregas-permeable pores. In one embodiment, the gas exchanger is a rigidsubstrate comprising a first surface, a second surface, and one or moreelastomeric pores. In one embodiment, the gas exchanger is agas-impermeable substrate comprising a first surface, a second surface,and a one or more gas-permeable pores between said first surface andsaid second surface. In one embodiment, the gas exchanger is a rigidsubstrate comprising a first surface, a second surface, and a one ormore elastomeric pores between said first surface and said secondsurface. In one embodiment, the gas exchanger is a gas-impermeablesubstrate comprising a first surface, a second surface, and one or moregas-permeable pores contacting at least one of said first surface andsaid second surface. In one embodiment, the gas exchanger is a rigidsubstrate comprising a first surface, a second surface, and one or moreelastomeric pores contacting at least one of said first surface and saidsecond surface.

In one embodiment, the gas exchanger is a gas-impermeable membranecomprising a first surface, a second surface, and one or moregas-permeable regions. In one embodiment, the gas exchanger is a rigidmembrane comprising a first surface, a second surface, and one or moreelastomeric regions. In one embodiment, the gas exchanger is agas-impermeable membrane comprising a first surface, a second surface,and a one or more gas-permeable regions between said first surface andsaid second surface. In one embodiment, the gas exchanger is a rigidmembrane comprising a first surface, a second surface, and a one or moreelastomeric regions between said first surface and said second surface.In one embodiment, the gas exchanger is a gas-impermeable membranecomprising a first surface, a second surface, and one or moregas-permeable regions contacting at least one of said first surface andsaid second surface. In one embodiment, the gas exchanger is a rigidmembrane comprising a first surface, a second surface, and one or moreelastomeric regions contacting at least one of said first surface andsaid second surface.

In one embodiment, the gas exchanger is a gas-impermeable sheetcomprising a first surface, a second surface, and one or moregas-permeable regions. In one embodiment, the gas exchanger is a rigidsheet comprising a first surface, a second surface, and one or moreelastomeric regions. In one embodiment, the gas exchanger is agas-impermeable sheet comprising a first surface, a second surface, anda one or more gas-permeable regions between said first surface and saidsecond surface. In one embodiment, the gas exchanger is a rigid sheetcomprising a first surface, a second surface, and a one or moreelastomeric regions between said first surface and said second surface.In one embodiment, the gas exchanger is a gas-impermeable sheetcomprising a first surface, a second surface, and one or moregas-permeable regions contacting at least one of said first surface andsaid second surface. In one embodiment, the gas exchanger is a rigidsheet comprising a first surface, a second surface, and one or moreelastomeric regions contacting at least one of said first surface andsaid second surface.

In one embodiment, the gas exchanger is a gas-impermeable substratecomprising a first surface, a second surface, and one or moregas-permeable film. In one embodiment, the gas exchanger is a rigid filmcomprising a first surface, a second surface, and one or moreelastomeric regions. In one embodiment, the gas exchanger is agas-impermeable film comprising a first surface, a second surface, and aone or more gas-permeable regions between said first surface and saidsecond surface. In one embodiment, the gas exchanger is a rigid filmcomprising a first surface, a second surface, and a one or moreelastomeric regions between said first surface and said second surface.In one embodiment, the gas exchanger is a gas-impermeable filmcomprising a first surface, a second surface, and one or moregas-permeable regions contacting at least one of said first surface andsaid second surface. In one embodiment, the gas exchanger is a rigidfilm comprising a first surface, a second surface, and one or moreelastomeric regions contacting at least one of said first surface andsaid second surface.

In one embodiment, the gas exchanger is a gas-impermeable membranecomprising a first surface, a second surface, and one or moregas-permeable pores. In one embodiment, the gas exchanger is a rigidmembrane comprising a first surface, a second surface, and one or moreelastomeric pores. In one embodiment, the gas exchanger is agas-impermeable membrane comprising a first surface, a second surface,and a one or more gas-permeable pores between said first surface andsaid second surface. In one embodiment, the gas exchanger is a rigidmembrane comprising a first surface, a second surface, and a one or moreelastomeric pores between said first surface and said second surface. Inone embodiment, the gas exchanger is a gas-impermeable membranecomprising a first surface, a second surface, and one or moregas-permeable pores contacting at least one of said first surface andsaid second surface. In one embodiment, the gas exchanger is a rigidmembrane comprising a first surface, a second surface, and one or moreelastomeric pores contacting at least one of said first surface and saidsecond surface.

In one embodiment, the gas exchanger is a gas-impermeable filmcomprising a first surface, a second surface, and one or moregas-permeable pores. In one embodiment, the gas exchanger is a rigidfilm comprising a first surface, a second surface, and one or moreelastomeric pores. In one embodiment, the gas exchanger is agas-impermeable film comprising a first surface, a second surface, and aone or more gas-permeable pores between said first surface and saidsecond surface. In one embodiment, the gas exchanger is a rigid filmcomprising a first surface, a second surface, and a one or moreelastomeric pores between said first surface and said second surface. Inone embodiment, the gas exchanger is a gas-impermeable film comprising afirst surface, a second surface, and one or more gas-permeable porescontacting at least one of said first surface and said second surface.In one embodiment, the gas exchanger is a rigid film comprising a firstsurface, a second surface, and one or more elastomeric pores contactingat least one of said first surface and said second surface.

In one embodiment, the gas exchanger is a gas-impermeable sheetcomprising a first surface, a second surface, and one or moregas-permeable pores. In one embodiment, the gas exchanger is a rigidsheet comprising a first surface, a second surface, and one or moreelastomeric pores. In one embodiment, the gas exchanger is agas-impermeable sheet comprising a first surface, a second surface, anda one or more gas-permeable pores between said first surface and saidsecond surface. In one embodiment, the gas exchanger is a rigid sheetcomprising a first surface, a second surface, and a one or moreelastomeric pores between said first surface and said second surface. Inone embodiment, the gas exchanger is a gas-impermeable sheet comprisinga first surface, a second surface, and one or more gas-permeable porescontacting at least one of said first surface and said second surface.In one embodiment, the gas exchanger is a rigid sheet comprising a firstsurface, a second surface, and one or more elastomeric pores contactingat least one of said first surface and said second surface.

In one embodiment, the gas exchanger is a gas-impermeable membranecomprising a first surface, a second surface, and one or moregas-permeable pores, wherein said membrane comprises cyclic olefincopolymer (COP) and said pores comprise polydimethylsiloxane (PDMS). Inone embodiment, the gas exchanger is a gas-impermeable membranecomprising a first surface, a second surface, and a one or moregas-permeable pores between said first surface and said second surface,wherein said membrane comprises cyclic olefin copolymer (COP) and saidpores comprise polydimethylsiloxane (PDMS). In one embodiment, the gasexchanger is a gas-impermeable membrane comprising a first surface, asecond surface, and one or more gas-permeable pores contacting at leastone of said first surface and said second surface, wherein said membranecomprises cyclic olefin copolymer (COP) and said pores comprisepolydimethylsiloxane (PDMS).

Furthermore, the gas exchanger may be coated with or have a film of aparticular material in order to enhance bonding. For example, he presentinvention contemplates, in one embodiment, a gas exchanger comprising aporous, gas-impermeable substrate may not only have the pores filledwith a gas-permeable material, but may also have a layer or coating orfilm of the gas-permeable material on top of it.

“Like dissolves like” is a common expression used by chemists toremember how some solvents interact with solutes. It refers to “polar”and “nonpolar” solvents and solutes. For example, water is polar and oilis non polar. Like does not dissolve like well, meaning that water willnot dissolve oil. For example, water is polar and salt (NaCl) is ionic(which is considered extremely polar). Like dissolves like, that meanspolar dissolves polar, so water dissolves salt. Much the same, “likebonds to like.” It has been found that materials bond more easily, suchas through chemical treatment, plasma treatment, etc. For example, PDMSbonds easily to PDMS as compared to other polymers. As such, in oneembodiment, the gas exchanger may have a coating, or film, or layer,which allows it to more easily bond to other structures.

In one embodiment, the gas exchanger is a gas-impermeable substratecomprising a first surface, a second surface, one or more gas-permeableregions, and a gas-permeable coating on said first surface. In oneembodiment, the gas exchanger is a gas-impermeable substrate comprisinga first surface, a second surface, a one or more gas-permeable regionsbetween said first surface and said second surface, and a gas-permeablecoating on said first surface. In one embodiment, the gas exchanger is agas-impermeable substrate comprising a first surface, a second surface,one or more gas-permeable regions contacting at least one of said firstsurface and said second surface, and a gas-permeable coating on saidfirst surface.

In one embodiment, the gas exchanger is a gas-impermeable substratecomprising a first surface, a second surface, one or more gas-permeableregions, and a gas-permeable coating on said first surface, wherein saidmembrane comprises cyclic olefin copolymer (COP) and said regions andcoating comprise polydimethylsiloxane (PDMS). In one embodiment, the gasexchanger is a gas-impermeable substrate comprising a first surface, asecond surface, a one or more gas-permeable regions between said firstsurface and said second surface, and a gas-permeable coating on saidfirst surface, wherein said membrane comprises cyclic olefin copolymer(COP) and said regions and coating comprise polydimethylsiloxane (PDMS).In one embodiment, the gas exchanger is a gas-impermeable substratecomprising a first surface, a second surface, one or more gas-permeableregions contacting at least one of said first surface and said secondsurface, and a gas-permeable coating on said first surface, wherein saidmembrane comprises cyclic olefin copolymer (COP) and said regions andcoating comprise polydimethylsiloxane (PDMS).

In one embodiment, the gas exchanger is a gas-impermeable membranecomprising a first surface, a second surface, one or more gas-permeableregions, and a gas-permeable coating on said first surface. In oneembodiment, the gas exchanger is a gas-impermeable membrane comprising afirst surface, a second surface, a one or more gas-permeable regionsbetween said first surface and said second surface, and a gas-permeablecoating on said first surface. In one embodiment, the gas exchanger is agas-impermeable membrane comprising a first surface, a second surface,one or more gas-permeable regions contacting at least one of said firstsurface and said second surface, and a gas-permeable coating on saidfirst surface.

In one embodiment, the gas exchanger is a gas-impermeable membranecomprising a first surface, a second surface, one or more gas-permeableregions, and a gas-permeable coating on said first surface, wherein saidmembrane comprises cyclic olefin copolymer (COP) and said regions andcoating comprise polydimethylsiloxane (PDMS). In one embodiment, the gasexchanger is a gas-impermeable membrane comprising a first surface, asecond surface, a one or more gas-permeable regions between said firstsurface and said second surface, and a gas-permeable coating on saidfirst surface, wherein said membrane comprises cyclic olefin copolymer(COP) and said regions and coating comprise polydimethylsiloxane (PDMS).In one embodiment, the gas exchanger is a gas-impermeable membranecomprising a first surface, a second surface, one or more gas-permeableregions contacting at least one of said first surface and said secondsurface, and a gas-permeable coating on said first surface, wherein saidmembrane comprises cyclic olefin copolymer (COP) and said regions andcoating comprise polydimethylsiloxane (PDMS).

In one embodiment, the gas exchanger is a gas-impermeable sheetcomprising a first surface, a second surface, one or more gas-permeableregions, and a gas-permeable coating on said first surface. In oneembodiment, the gas exchanger is a gas-impermeable sheet comprising afirst surface, a second surface, a one or more gas-permeable regionsbetween said first surface and said second surface, and a gas-permeablecoating on said first surface. In one embodiment, the gas exchanger is agas-impermeable sheet comprising a first surface, a second surface, oneor more gas-permeable regions contacting at least one of said firstsurface and said second surface, and a gas-permeable coating on saidfirst surface.

In one embodiment, the gas exchanger is a gas-impermeable sheetcomprising a first surface, a second surface, one or more gas-permeableregions, and a gas-permeable coating on said first surface, wherein saidmembrane comprises cyclic olefin copolymer (COP) and said regions andcoating comprise polydimethylsiloxane (PDMS). In one embodiment, the gasexchanger is a gas-impermeable sheet comprising a first surface, asecond surface, a one or more gas-permeable regions between said firstsurface and said second surface, and a gas-permeable coating on saidfirst surface, wherein said membrane comprises cyclic olefin copolymer(COP) and said regions and coating comprise polydimethylsiloxane (PDMS).In one embodiment, the gas exchanger is a gas-impermeable sheetcomprising a first surface, a second surface, one or more gas-permeableregions contacting at least one of said first surface and said secondsurface, and a gas-permeable coating on said first surface, wherein saidmembrane comprises cyclic olefin copolymer (COP) and said regions andcoating comprise polydimethylsiloxane (PDMS).

In one embodiment, the gas exchanger is a gas-impermeable filmcomprising a first surface, a second surface, one or more gas-permeableregions, and a gas-permeable coating on said first surface. In oneembodiment, the gas exchanger is a gas-impermeable film comprising afirst surface, a second surface, a one or more gas-permeable regionsbetween said first surface and said second surface, and a gas-permeablecoating on said first surface. In one embodiment, the gas exchanger is agas-impermeable film comprising a first surface, a second surface, oneor more gas-permeable regions contacting at least one of said firstsurface and said second surface, and a gas-permeable coating on saidfirst surface.

In one embodiment, the gas exchanger is a gas-impermeable filmcomprising a first surface, a second surface, one or more gas-permeableregions, and a gas-permeable coating on said first surface, wherein saidmembrane comprises cyclic olefin copolymer (COP) and said regions andcoating comprise polydimethylsiloxane (PDMS). In one embodiment, the gasexchanger is a gas-impermeable film comprising a first surface, a secondsurface, a one or more gas-permeable regions between said first surfaceand said second surface, and a gas-permeable coating on said firstsurface, wherein said membrane comprises cyclic olefin copolymer (COP)and said regions and coating comprise polydimethylsiloxane (PDMS). Inone embodiment, the gas exchanger is a gas-impermeable film comprising afirst surface, a second surface, one or more gas-permeable regionscontacting at least one of said first surface and said second surface,and a gas-permeable coating on said first surface, wherein said membranecomprises cyclic olefin copolymer (COP) and said regions and coatingcomprise polydimethylsiloxane (PDMS).

In one embodiment, the gas exchanger is a gas-impermeable substratecomprising a first surface, a second surface, one or more gas-permeablepores, and a gas-permeable coating on said first surface. In oneembodiment, the gas exchanger is a gas-impermeable substrate comprisinga first surface, a second surface, a one or more gas-permeable poresbetween said first surface and said second surface, and a gas-permeablecoating on said first surface. In one embodiment, the gas exchanger is agas-impermeable substrate comprising a first surface, a second surface,one or more gas-permeable pores contacting at least one of said firstsurface and said second surface, and a gas-permeable coating on saidfirst surface.

In one embodiment, the gas exchanger is a gas-impermeable substratecomprising a first surface, a second surface, one or more gas-permeablepores, and a gas-permeable coating on said first surface, wherein saidmembrane comprises cyclic olefin copolymer (COP) and said pores andcoating comprise polydimethylsiloxane (PDMS). In one embodiment, the gasexchanger is a gas-impermeable substrate comprising a first surface, asecond surface, a one or more gas-permeable pores between said firstsurface and said second surface, and a gas-permeable coating on saidfirst surface, wherein said membrane comprises cyclic olefin copolymer(COP) and said pores and coating comprise polydimethylsiloxane (PDMS).In one embodiment, the gas exchanger is a gas-impermeable substratecomprising a first surface, a second surface, one or more gas-permeablepores contacting at least one of said first surface and said secondsurface, and a gas-permeable coating on said first surface, wherein saidmembrane comprises cyclic olefin copolymer (COP) and said pores andcoating comprise polydimethylsiloxane (PDMS).

In one embodiment, the gas exchanger is a gas-impermeable membranecomprising a first surface, a second surface, one or more gas-permeablepores, and a gas-permeable coating on said first surface. In oneembodiment, the gas exchanger is a gas-impermeable membrane comprising afirst surface, a second surface, a one or more gas-permeable poresbetween said first surface and said second surface, and a gas-permeablecoating on said first surface. In one embodiment, the gas exchanger is agas-impermeable membrane comprising a first surface, a second surface,one or more gas-permeable pores contacting at least one of said firstsurface and said second surface, and a gas-permeable coating on saidfirst surface.

In one embodiment, the gas exchanger is a gas-impermeable membranecomprising a first surface, a second surface, one or more gas-permeablepores, and a gas-permeable coating on said first surface, wherein saidmembrane comprises cyclic olefin copolymer (COP) and said pores andcoating comprise polydimethylsiloxane (PDMS). In one embodiment, the gasexchanger is a gas-impermeable membrane comprising a first surface, asecond surface, a one or more gas-permeable pores between said firstsurface and said second surface, and a gas-permeable coating on saidfirst surface, wherein said membrane comprises cyclic olefin copolymer(COP) and said pores and coating comprise polydimethylsiloxane (PDMS).In one embodiment, the gas exchanger is a gas-impermeable membranecomprising a first surface, a second surface, one or more gas-permeablepores contacting at least one of said first surface and said secondsurface, and a gas-permeable coating on said first surface, wherein saidmembrane comprises cyclic olefin copolymer (COP) and said pores andcoating comprise polydimethylsiloxane (PDMS).

In one embodiment, the gas exchanger is a gas-impermeable sheetcomprising a first surface, a second surface, one or more gas-permeablepores, and a gas-permeable coating on said first surface. In oneembodiment, the gas exchanger is a gas-impermeable sheet comprising afirst surface, a second surface, a one or more gas-permeable poresbetween said first surface and said second surface, and a gas-permeablecoating on said first surface. In one embodiment, the gas exchanger is agas-impermeable sheet comprising a first surface, a second surface, oneor more gas-permeable pores contacting at least one of said firstsurface and said second surface, and a gas-permeable coating on saidfirst surface.

In one embodiment, the gas exchanger is a gas-impermeable sheetcomprising a first surface, a second surface, one or more gas-permeablepores, and a gas-permeable coating on said first surface, wherein saidmembrane comprises cyclic olefin copolymer (COP) and said pores andcoating comprise polydimethylsiloxane (PDMS). In one embodiment, the gasexchanger is a gas-impermeable sheet comprising a first surface, asecond surface, a one or more gas-permeable pores between said firstsurface and said second surface, and a gas-permeable coating on saidfirst surface, wherein said membrane comprises cyclic olefin copolymer(COP) and said pores and coating comprise polydimethylsiloxane (PDMS).In one embodiment, the gas exchanger is a gas-impermeable sheetcomprising a first surface, a second surface, one or more gas-permeablepores contacting at least one of said first surface and said secondsurface, and a gas-permeable coating on said first surface, wherein saidmembrane comprises cyclic olefin copolymer (COP) and said pores andcoating comprise polydimethylsiloxane (PDMS).

In one embodiment, the gas exchanger is a gas-impermeable filmcomprising a first surface, a second surface, one or more gas-permeablepores, and a gas-permeable coating on said first surface. In oneembodiment, the gas exchanger is a gas-impermeable film comprising afirst surface, a second surface, a one or more gas-permeable poresbetween said first surface and said second surface, and a gas-permeablecoating on said first surface. In one embodiment, the gas exchanger is agas-impermeable film comprising a first surface, a second surface, oneor more gas-permeable pores contacting at least one of said firstsurface and said second surface, and a gas-permeable coating on saidfirst surface.

In one embodiment, the gas exchanger is a gas-impermeable filmcomprising a first surface, a second surface, one or more gas-permeablepores, and a gas-permeable coating on said first surface, wherein saidmembrane comprises cyclic olefin copolymer (COP) and said pores andcoating comprise polydimethylsiloxane (PDMS). In one embodiment, the gasexchanger is a gas-impermeable film comprising a first surface, a secondsurface, a one or more gas-permeable pores between said first surfaceand said second surface, and a gas-permeable coating on said firstsurface, wherein said membrane comprises cyclic olefin copolymer (COP)and said pores and coating comprise polydimethylsiloxane (PDMS). In oneembodiment, the gas exchanger is a gas-impermeable film comprising afirst surface, a second surface, one or more gas-permeable porescontacting at least one of said first surface and said second surface,and a gas-permeable coating on said first surface, wherein said membranecomprises cyclic olefin copolymer (COP) and said pores and coatingcomprise polydimethylsiloxane (PDMS).

One embodiment of the present invention is a fluidic device formonitoring biological function is contemplated, said fluidic devicecomprising (i) a first channel, (ii) a second channel, (iii) a membranedisposed between said first channel and second channel, and (iv) a gasexchanger contacting at least one of said first and second channelconfigured to be able to control the rate of gas transport into saidfluidic device. In one embodiment, said fluidic device is a microfluidicdevice. In one embodiment, the first and second channel layers are gasimpermeable. In one embodiment, said first and second channel layers areresistant to absorption of small molecules. In one embodiment, at leastone of said first and second channel layers comprise (cyclic olefincopolymer) COP. In one embodiment, at least one of said first and secondchannels comprise cells. In one embodiment, said cells are human cells.In one embodiment, said gas exchanger provides mechanical stability tosaid fluidic device. In one embodiment, said gas exchanger at leastpartially encloses at least one of said first channel or said secondchannel. In one embodiment, said gas exchanger at least partiallyborders at least one of said first channel or said second channel. Inone embodiment, said gas exchanger comprises two polymer layers. In oneembodiment, said gas exchanger comprises polymethylpentene (PMP). In oneembodiment, said gas exchanger comprises polydimethylsiloxane (PDMS). Inone embodiment, said gas exchanger comprises polyethylene terephthalate(PET). In one embodiment, said gas exchanger comprisespolytetrafluoroethene (PTFE or Teflon). In one embodiment, said gasexchanger comprisespoly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene](TeflonAF2400). In one embodiment, said gas exchanger comprises apolymer film comprising a plurality of pores, said plurality of poresdefining a porosity. In one embodiment, said porosity is between 0.05%and 15%. In one embodiment, said porosity regulates a rate of gastransport. In one embodiment, said plurality pores are filled with agas-permeable polymer. In one embodiment, said gas-permeable polymercomprises polydimethylsiloxane (PDMS). In one embodiment, said polymerfilm is polyethylene terephthalate (PET). In one embodiment, said gasexchanger comprises a gas impermeable film comprising a plurality ofgas-permeable pores, said plurality of pores defining a porosity. In oneembodiment, said gas exchanger is less gas-permeable thanpolydimethylsiloxane (PDMS). In one embodiment, said gas-exchangercomprises less than 0.025-1 μL by volume of porosity. In one embodiment,said gas exchanger runs along the length of at least one of said firstchannel or said second channel. In one embodiment, said gas exchanger isconfigured for providing a constant rate of gas transport along thelength of at least one of said first channel and said second channel. Inone embodiment, said membrane comprises a gas-permeable polymer. In oneembodiment, said membrane comprises polydimethylsiloxane (PDMS). In oneembodiment, said membrane comprises a plurality of pores, said pluralityof pores defining a porosity. In one embodiment, said porosity of saidmembrane is between 5% and 10%. In one embodiment, said porosityregulates a rate of gas transport through the membrane. In oneembodiment, said device further comprises one or more sensors. In oneembodiment, at least one sensor is an oxygen sensor. In one embodiment,said fluidic device comprises a hypoxic environment in said at least oneof said first and second channels.

In one embodiment, a fluidic device is contemplated for monitoringbiological function, said fluidic device comprising (i) a first channellayer including a first channel, (ii) a second channel layer including asecond channel, (iii) a membrane located between said first channellayer and second channel layer, and (iv) a gas exchanger contacting atleast one of said first and second channels configured to be able tointroduce gas flow into said fluidic device. In one embodiment, saidfluidic device is a microfluidic device. In one embodiment, the firstand second channel layers are gas impermeable. In one embodiment, saidfirst and second channel layers are resistant to absorption of smallmolecules. In one embodiment, at least one of said first and secondchannel layers comprise (cyclic olefin copolymer) COP. In oneembodiment, at least one of said first and second channels comprisecells. In one embodiment, said cells are human cells. In one embodiment,said gas exchanger provides mechanical stability to said fluidic device.In one embodiment, said gas exchanger at least partially encloses atleast one of said first channel or said second channel. In oneembodiment, said gas exchanger at least partially borders at least oneof said first channel or said second channel. In one embodiment, saidgas exchanger comprises two polymer layers. In one embodiment, said gasexchanger comprises polymethylpentene (PMP). In one embodiment, said gasexchanger comprises polydimethylsiloxane (PDMS). In one embodiment, saidgas exchanger comprises polyethylene terephthalate (PET). In oneembodiment, said gas exchanger comprises polytetrafluoroethene (PTFE orTeflon). In one embodiment, said gas exchanger comprisespoly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene](TeflonAF2400). In one embodiment, said gas exchanger comprises apolymer film comprising a plurality of pores, said plurality of poresdefining a porosity. In one embodiment, said porosity is between 0.05%and 15%. In one embodiment, said porosity regulates a rate of gastransport. In one embodiment, said plurality pores are filled with agas-permeable polymer. In one embodiment, said gas-permeable polymercomprises polydimethylsiloxane (PDMS). In one embodiment, said polymerfilm is polyethylene terephthalate (PET). In one embodiment, said gasexchanger comprises a gas impermeable film comprising a plurality ofgas-permeable pores, said plurality of pores defining a porosity. In oneembodiment, said gas exchanger is less gas-permeable thanpolydimethylsiloxane (PDMS). In one embodiment, said gas-exchangercomprises less than 0.025-1 μL by volume of porosity. In one embodiment,said gas exchanger runs along the length of at least one of said firstchannel or said second channel. In one embodiment, said gas exchanger isconfigured for providing a constant rate of gas transport along thelength of at least one of said first channel and said second channel. Inone embodiment, said membrane comprises a gas-permeable polymer. In oneembodiment, said membrane comprises polydimethylsiloxane (PDMS). In oneembodiment, said membrane comprises a plurality of pores, said pluralityof pores defining a porosity. In one embodiment, said porosity of saidmembrane is between 5% and 10%. In one embodiment, said porosityregulates a rate of gas transport through the membrane. In oneembodiment, the method further comprises one or more sensors. In oneembodiment, at least one sensor is an oxygen sensor. In one embodiment,said fluidic device comprises a hypoxic environment in said at least oneof said first and second channels.

Another embodiment of the present invention is an upgraded perfusionmanifold assembly that minimizes the amount of small molecule compoundto absorb into its materials. In one embodiment, the perfusion manifoldassembly comprises i) a cover or lid assembly configured to serve as thetop of ii) one or more fluid reservoirs, iii) a gasketing layer undersaid fluid reservoir(s), iv) a fluidic backplane under, and in fluidiccommunication with, said fluid reservoirs, v) a capping layer over saidfluidic backplane, and vi) a projecting member or skirt for engaging themicrofluidic device or a carrier containing a microfluidic device.

One embodiment of the present invention is a method of fabricating amicrofluidic device, comprising: a) providing a microfluidic devicecomprising a channel; b) selecting a gas exchanger of a porosity,wherein said porosity determines a rate of gas transport; and c) cappingsaid channel with said gas exchanger. In one embodiment, said channelcomprises a first chamber and a second chamber separated by a membrane.In one embodiment, said microfluidic device is able to maintain aconstant rate of gas transport with and without fluid flow. In oneembodiment, said gas comprises oxygen. In one embodiment, said gasexchanger comprises polyethylene terephthalate (PET). In one embodiment,said gas exchanger comprises polydimethylsiloxane (PDMS). In oneembodiment, wherein said gas exchanger is a device comprising a firstgas impermeable substrate, said first gas impermeable substrate having(i) a first side, (ii) a second side, and (iii) one or moregas-permeable regions are between said first side and said second side.In one embodiment, said gas exchanger is a film. In one embodiment, saidregions are pores. In one embodiment, said regions contact at least oneof said first side and said second side. In one embodiment, said channelis an open channel. In one embodiment, said gas exchanger comprisespores filled with a gas-permeable material, said pores defining saidporosity. In one embodiment, said gas exchanger comprises gasimpermeable regions and gas-permeable regions, wherein saidgas-permeable regions represent less than 10% by volume of the gasexchanger. In one embodiment, said microfluidic device further comprisescells, and said rate of gas transport is selected to maintain theviability of said cells. In one embodiment, said rate of gas transportcreates a gas concentration profile within said microfluidic device. Inone embodiment, said microfluidic device further comprises liver cells,and said gas concentration profile is liver oxygen zonation. In oneembodiment, said microfluidic device further comprises cancer cells, andsaid gas concentration profile is a hypoxic environment. In oneembodiment, said microfluidic device further comprises colon cells, andsaid gas concentration profile is a hypoxic lumen environment. In oneembodiment, said gas exchanger limits the flow of gas into themicrofluidic device. In one embodiment, said gas exchanger increases theflow of gas into the microfluidic device.

One embodiment of the present invention is a fluidic device comprising afirst substrate having an open channel and a second substrate comprisinga gas exchanger, wherein said second substrate caps the first substrateforming an at least partially enclosed channel. In one embodiment, saidgas exchanger comprises polyethylene terephthalate (PET). In oneembodiment, said gas exchanger comprises polydimethylsiloxane (PDMS). Inone embodiment, said gas exchanger is a device comprising a gasimpermeable material, said gas impermeable material having (i) a firstside, (ii) a second side, and (iii) one or more gas-permeable regionsare between said first side and said second side. In one embodiment,said material is a film. In one embodiment, said regions are pores. Inone embodiment, said regions contact at least one of said first side andsaid second side. In one embodiment, said regions represent less than10% by volume of the gas exchanger. In one embodiment, said device is amicrofluidic device. In one embodiment, said gas exchanger comprises acomposite of a gas-permeable material and a gas-impermeable material. Inone embodiment, said gas exchanger comprises a first gas-permeablesubstrate and a second gas-impermeable substrate.

One embodiment of the present invention is a method of controlling gastransport, comprising: a) providing a fluidic device comprising body anda gas exchanger contacting said body, said gas exchanger comprising agas-impermeable polymer substrate with gas-permeable regions, saidsubstrate comprising first and second sides, said regions creating aporosity; and b) introducing gas on said first side of said substrate,wherein the rate of gas transport to said second side is controlled bysaid porosity. In one embodiment, said polymer comprises polyethyleneterephthalate (PET) and said regions comprises polydimethylsiloxane(PDMS). In one embodiment, said fluidic device body is gas-permeable. Inone embodiment, said fluidic device body is gas-impermeable. In oneembodiment, said gas exchanger comprises a gas-permeable polymer layerin contact with said first side of said substrate. In one embodiment,said fluidic device comprises at least one channel. In one embodiment,said gas exchanger comprises at least one wall of said at least onechannel. In one embodiment, said fluidic device contains cells. In oneembodiment, said gas comprises oxygen. In one embodiment, said gasexchanger reduces the rate of gas transport into said microfluidicdevice. In one embodiment, said gas exchanger increases the rate of gastransport into said microfluidic device. In one embodiment, said gasexchanger maintains a rate of gas transport into said microfluidicdevice with and without fluid flow in said channel. In one embodiment,said gas exchanger is selected from the list comprising a film, a sheet,a composite, a gas-exchange membrane, a lamination, a pore-filledsubstrate, a pore-filled film, a pore-filled membrane, and a pore-filledcomposite. In one embodiment, said regions are selected from the listcomprising pores, conduits, indentations, holes, and channels. In oneembodiment, said regions contact at least one of said first side andsaid second side. In one embodiment, said regions represent less than10% by volume of the gas exchanger.

One embodiment of the present invention is a method of fabricating a gasexchanger comprising: a) providing a gas impermeable polymer substratewith pores, said substrate comprising first and second surfaces, saidpores creating a porosity; b) coating said first surface with an uncuredgas-permeable polymer, such that said uncured gas-permeable polymerpenetrates said pores; c) removing excess uncured polymer from saidfirst and second surfaces, such that said first and second surfaces aresubstantially free of said uncured polymer, while said pores are filledwith said uncured gas-permeable polymer; and d) curing said uncuredgas-permeable polymer in said pores to fabricate a substantially gasimpermeable gas exchanger. One embodiment of the present invention is amicrofluidic device comprising said fabricated gas exchanger. In oneembodiment, said gas-impermeable polymer substrate comprisespolyethylene terephthalate (PET). In one embodiment, said gas-permeablepolymer comprises polydimethylsiloxane (PDMS). In one embodiment, saidpores comprises less than 100 μL by volume of gas-permeable polymer. Inone embodiment, said pores comprises less than 50 μL by volume of saidgas-permeable polymer. In one embodiment, said pores comprises less than10 μL by volume of said gas-permeable polymer. In one embodiment, saidpores comprises less than 1 μL by volume of said gas-permeable polymer.In one embodiment, the method further comprises the step of degassingsaid uncured gas-permeable polymer.

One embodiment of the present invention is a method of fabricating a gasexchanger comprising: a) providing (i) a first gas-impermeable polymersubstrate with pores, said substrate comprising first and secondsurfaces, said pores creating a porosity, and (ii) a secondgas-permeable polymer substrate; and b) laminating said first surfacewith said second gas-permeable polymer substrate, such that saidgas-permeable polymer substrate covers said pores. In one embodiment,said first gas-impermeable polymer substrate comprises polyethyleneterephthalate (PET). In one embodiment, said second gas-permeablepolymer substrate comprises polydimethylsiloxane (PDMS). In oneembodiment, said first or second substrate is a film. In one embodiment,first or second substrate is a membrane.

One embodiment of the present invention is a method of fabricating a gasexchanger comprising: a) providing (i) a first gas-impermeable polymersubstrate with pores, said substrate comprising first and secondsurfaces, said pores creating a porosity, and (ii) a secondgas-permeable polymer substrate; and b) contacting said first surfacewith said second gas-permeable polymer substrate, such that saidgas-permeable polymer substrate forms to said gas-impermeable polymersubstrate, covering said pores. In one embodiment, said firstgas-impermeable polymer substrate comprises polyethylene terephthalate(PET). In one embodiment, said second gas-permeable polymer substratecomprises polydimethylsiloxane (PDMS). In one embodiment, said first orsecond substrate is a film. In one embodiment, said first or secondsubstrate is a membrane.

The present invention contemplates, in one embodiment, a method ofcontrolling gas transport, comprising: a) providing a substantiallygas-impermeable microfluidic device comprising i) a gas exchanger andii) living cells in a channel or chamber, said device comprising a rigidpolymer; and b) introducing culture media into said channel or chamberat a flow rate, said culture media carrying gas, wherein the rate of gastransport to said living cells is controlled by said gas exchanger. Inone embodiment, said rigid polymer is polycarbonate. In one embodiment,said rigid polymer has a modulus of elasticity between 0.1 and 150 GPa.In one embodiment, said gas exchanger comprises a film ofpolydimethylsiloxane (PDMS) positioned below said channel or chamber. Inone embodiment, said gas exchanger comprises a film of a non-permeablepolymer with gas-permeable pores, said film positioned below saidchannel or chamber.

The present invention contemplates, in one embodiment, a method ofcontrolling gas transport in a microfluidic device, comprising: a)providing a substantially gas-impermeable microfluidic device comprisinga plurality of outer sides comprising substantially gas-impermeablepolymer having a modulus of elasticity between 0.1 and 150 GPa, and asubstantially gas-permeable inner membrane disposed between a firstchannel and a second channel; and b) introducing a fluid into said atleast one of said first channel or said second channel at a flow rate,wherein said substantially gas-permeable inner membrane is configured toallow gas transport between said first channel and said second channel.

In one embodiment, a microfluidic device is contemplated comprising aplurality of outer sides comprising substantially gas-impermeablepolymer having a modulus of elasticity between 0.1 and 150 GPa, and asubstantially gas-permeable inner membrane. In one embodiment, saidsubstantially gas-permeable inner membrane comprisespolydimethylsiloxane (PDMS). In one embodiment, wherein saidpolydimethylsiloxane (PDMS) membrane is configured for stretching.

In one embodiment, a method is contemplated comprising: a) providing amicrofluidic device comprising a plurality of outer sides comprisingsubstantially gas-impermeable polymer having a modulus of elasticitybetween 0.1 and 150 GPa, and a substantially gas-permeable innermembrane; and b) stretching said membrane. In one embodiment, whereinsaid substantially gas-permeable inner membrane comprisespolydimethylsiloxane (PDMS). In one embodiment, wherein said stretchingis achieved by applying differential pressure across said membrane. Inone embodiment, wherein said microfluidic device further comprises a gasexchanger.

In one embodiment, a microfluidic device is contemplated comprising: (i)a first channel and a second channel, each of said first channel andsecond channel comprising a plurality of walls, wherein at least one ofsaid walls are gas-permeable having a modulus of elasticity below 0.1GPa and at least one of said walls are gas-impermeable having a modulusof elasticity between 0.1 and 150 GPa, and (ii) a gas-permeable membranedisposed between said first and second channel, said membrane having amodulus of elasticity less than 0.1 GPa.

Another embodiment of the present invention is applying the presentlydescribed embodiments gas exchangers or gas transport membranes to anymicrofluidic cell culture system. It may be desirable to better controlthe gas exchange into various microfluidic devices on the marketdepending on the unique needs of the scientist. Oxygen delivery to thecells in microfluidic culture is an industry-wide problem, particularlyas manufacturers adopt thermoplastics due to their easier manufacturingand/or lower gas absorption. The idea of replacing one of the walls(e.g. the device's bottom) with a gas exchanger as described herein is abrilliant solution to this major problem.

For example, the embodiments of gas exchangers described herein may beapplied to the Mimetas's microfluidic device of U.S. Pat. No.10,532,355, which is incorporated by reference herein in its entirety.The bottom of the Mimeta's microfluidic products such as theOrganoPlate® 2-lane, OrganoPlate® 3-lane, etc. may be replaced with agas exchanger in order to achieve better control over the gasconcentrations within the microfluidic device. The gas exchanger mayreplace any structural elements of the microfluidic device. Inparticular, the gas exchanger may replace structural elements in contactwith channels within the microfluidic device. In cases wherein themicrofluidic device has more than one channel, the gas exchanger may beplaced between the channels of the microfluidic device. Other exemplaryembodiments of microfluidic devices that may be advantageously combinedwith the microfluidic devices of Aim Biotech (e.g. U.S. PatentPublication No. 20180327700A1) and the microfluidic devices of theMassachusetts Institute of Technology's Mechanobiology Lab (e.g. U.S.Pat. Nos. 9,121,847 and 9,261,496). These three patent applications areincorporated in their entirety herein.

In one embodiment, a method is contemplated, comprising: (i) providing amicrofluidic device and a gas exchanger; and (ii) applying said gasexchanger to said microfluidic device, altering the gas-permeability ofsaid microfluidic device. A method is contemplated, comprising: (i)providing a microfluidic device and a gas exchanger; (ii) removing asubstrate of said microfluidic device; and (iii) replacing saidsubstrate with said gas exchanger, altering the gas-permeability of saidmicrofluidic device. A method is contemplated, comprising: (i) providinga microfluidic device comprising at least one channel and a membranewithin said channel; and (ii) applying a gas exchanger to said at leastone channel. In one embodiment, said channel comprises cells.

It is not intended that the microfluidic device be limited by the numberof channels; the microfluidic device to have a gas exchanger appliedthereto may have one, two, three, four, five, six, etc. channels.

Another embodiment of the present invention is an upgraded perfusionmanifold assembly that minimizes the amount of small molecule compoundto absorb into its materials. In one embodiment, the perfusion manifoldassembly comprises i) a cover or lid configured to serve as the top ofii) one or more fluid reservoirs, iii) a gasketing layer under saidfluid reservoir(s), iv) a fluidic backplane under, and in fluidiccommunication with, said fluid reservoirs, v) a capping layer over saidfluidic backplane, and vi) a projecting member or skirt for engaging themicrofluidic device or a carrier containing a microfluidic device.

The cover or lid assembly may aid in protecting the reservoirs from bothspilling and contamination. In one embodiment, the lid assemblycomprises a lid, filter(s), and a lid gasket. Filters may be configuredinto the lid assembly in order to aid in sterility of the fluid withinthe reservoirs. In one embodiment the filters are flat filters. Thesethin filters may be cut from a flat substrate material. In oneembodiment the filters are thick filters. These thick filters may be cutfrom a thick substrate material. In the embodiment wherein, the lidassembly comprises a lid gasket, the lid gasket may take on a variety ofembodiments. In one embodiment, the lid gasket is compressible. In oneembodiment, the lid gasket is adhesive. The lid gasket may vary inthickness in order to best seal the reservoirs off from the externalenvironment. Alternatively, in other embodiment, the lid gasketcomprises the filters, instead of having separate filters. In oneembodiment, the lid gasket is porous. In another embodiment the lidgasket is non-porous. In one embodiment, the lid gasket permanentlyconforms to the shape of the reservoirs after the first time thereservoirs is pressed into it. In another embodiment the lid gaskettemporarily conforms to the shape of the reservoirs after each time thelid gasket is pressed onto them. In yet another embodiment, the lidgasket does not conform to the shape of the reservoirs. The cover or lidassembly can be removed and the perfusion manifold assembly can still beused. In one embodiment, the lid assembly is held onto the reservoirusing a radial seal. An applied pressure is not necessarily required tocreate a seal. In another embodiment, the lid assembly is held onto thereservoir using one or more clips, screws or other retention mechanisms.

The fluid backplane may be used to route fluid from the reservoirs tothe microfluidic devices, such as a microfluidic device. In oneembodiment, the assembly further comprises fluid ports positioned at thebottom of the fluidic backplane. In one embodiment the fluidic backplanecomprises one or more fluidic resistors. Without being bound by theoryof any particular mechanism, it is believed that these resistors serveto stabilize the flow of fluid coming from the reservoirs so that astable flow can be delivered to the microfluidic device, and/or theyserve to provide a means for translating reservoir pressure to perfusionflow rate.

In previous renditions of this invention there has been a single layerresponsible for both capping and gasketing. The invention presented heresuggests two separate layers, i.e. one for gasketing and one for cappingthe fluidic backplane. In one embodiment both the fluid reservoirs andfluid backplane are fabricated from hard plastics, and as such may needa compressible gasket between them to protect from leaks at the sites offluid connections. Having two separate layers is advantageous as cappingthe fluidic backplane and gasketing between the fluidic backplane andreservoirs may be decoupled. Oftentimes materials having thecharacteristics necessary to be used as gaskets, especially transparentgaskets, have absorbency issues. By decoupling the functions of theprevious single layer, the amount of absorbing material may be minimizedin the perfusion manifold assembly and segregated/isolated to the layerresponsible for gasketing. In one embodiment both the capping andgasketing layers are transparent. It may advantageous to havetransparent capping and gasketing layers so that the fluidic backplanemay be imaged on a microscope if necessary. In one embodiment of the newinvention, the gasketing layer is made up of a compressible material,such as SEBS, while the capping layer is made up of an incompressiblematerial, such as COP. In another embodiment, the gasketing layer madeup of a compressible material may be coated with a thin layer of anincompressible material, such as with parylene, in order to make itnon-absorbent while still maintaining bulk flexibility and, therefore,the ability to seal or gasket the fluid layer to the reservoirs. Thecapping layer may be partially or completely coated in Parylene. In anexemplary embodiment, a partially coated capping layer fabricated out ofCOP is used in conjunction with a gasketing layer fabricated out ofSEBS. The combination of a partially Parylene-coated COP capping layerand SEBS gasketing layer is advantageous over a single, completelyParylene coated COP layer. Parylene is difficult to bond, whereas COPbonds well to other materials, including other parts made out of COP. Byusing two layers, one may seal the fluidic backplane to theParylene-coated COP capping layer by material bonding, and seal thecapping layer to the reservoirs with the SEBS gasketing layer. Further,when using two layers only a small piece of SEBS needs to be coated withParylene to successfully prevent absorption. If a single layer is used,any fluid-contacting surface may need to be coated with Parylene, whichmeans that the ports, the face of the components being sealed (such asthe reservoirs), and the entire length of the fluidic routing channelsin the perfusion manifold assembly would need to be coated. Coating thatmuch of the COP capping layer is difficult. When Parylene is coated, thepart needs to be held somewhere, much like Achille's heel.

In one embodiment the perfusion manifold assembly comprises a projectingmember or skirt. In one embodiment, the projecting member or skirt isengaged with a microfluidic device. In one embodiment, the microfluidicdevice comprises a first channel, a second channel and a membraneseparating at least a portion of said first and second channels. Inanother embodiment, wherein the microfluidic device is orientedvertically, the microfluidic device comprises a top channel, a bottomchannel, and a membrane separating at least a portion of said topchannel and bottom channel. In one embodiment, the microfluidic devicecomprises cells on the membrane and/or in or on the channels. Theprojecting member or skirt may be designed so that the fluidic backplaneis able to easily align with a connecting microfluidic device. In oneembodiment, the projecting member or skirt may be designed in order tointeract with a culture system.

The perfusion manifold assembly may be attached together via severalmethods. In one embodiment, screws may be used to secure the perfusionmanifold assembly. In another embodiment, clips are used to secure theperfusion manifold assembly. In another embodiment, adhesives are usedto secure the perfusion manifold assembly. In another embodiment,surface modification is used to secure the perfusion manifold assembly.In one embodiment, the perfusion manifold assembly is permanently bondedtogether. In one embodiment, the perfusion manifold assembly istemporarily bonded together.

If these above described perfusion manifold assemblies are to be usedwith low-absorbing, gas-impermeable microfluidic devices, the design ofthe perfusion manifold assembly may be used to introduce a desired gasconcentration into the microfluidic device. In one previously describedembodiment, the dissolved gas content of the media may be increasedprior to entering the microfluidic device by pressurizing the media withthe desired gas or by adding a carrier of the desired gas to the media.In one previously described embodiment, the concentration of oxygenwithin a media may be increased by pressurizing the media with aconcentration of oxygen or by increasing the concentration of oxygen inthe pressurized gas mixture or by adding an oxygen carrier, such ashemoglobin or hemocyanin, to the media. In one previously describedembodiment, the gas pressurizing the media may be a gas blanket. In oneembodiment, the dissolved gas content of the media in the perfusionmanifold assembly reservoirs may be increased prior to entering themicrofluidic device by pressurizing the media in the reservoirs with thedesired gas or a carrier of the gas in the headspace of the reservoir.In one embodiment, the concentration of oxygen within the media in theperfusion manifold assembly reservoirs may be increased by pressurizingthe media in the headspace of the reservoirs with oxygen or by adding anoxygen carrier, such as hemoglobin, to the media.

A perfusion manifold assembly is contemplated comprising (i) a lidconfigured to serve as the top of (ii) one or more fluid reservoirs,(iii) a gasketing layer resistant to absorption of small molecules undersaid fluid reservoir(s), (iv) a fluidic backplane under, and in fluidiccommunication with, said fluid reservoirs, (v) a capping layer resistantto the absorption of small molecules over said fluidic backplane, and(vi) a projecting member for engaging the microfluidic device. In oneembodiment, said gasketing layer comprises parylene-coated SEBS. In oneembodiment, said capping layer is fabricated from COP.

In both in vitro and in vivo experiments, researchers should considercompound distribution within the biological model and experimentalsetup, as distribution determines exposure—the concentration of compoundthat cells truly experience. Volume of distribution is typicallyassessed and accounted for in in vivo studies, but the effects of systemcomponents such as infusion tubing, syringes, tissue-culture plates andpipette tips are often missed. While absorption is a major component oferroneous compound distribution, undesired compound distribution alsoincludes compound pooling in one region of an experimental setup,adsorption, and other situations where a compound is not where ittheoretically should be or where the user would like it to be. Forexample, erroneous compound distribution may be when a compound is notuniformly distributed throughout a system. For example, erroneouscompound distribution may be when cells in the former portion of a cellculture channel metabolize the compound, such that cells in the latterportion of a cell culture channel are unable to be contacted by thecompound.

With microfluidic device experiments, compound distribution may beaddressed in a number of ways. Several of these are captured herein,where experimental conditions have been selected to optimize compoundexposure. Additionally, a compound distribution kit has been developeddirectly evaluate distribution and compound exposure. In analogy with invivo distribution studies, it may be recommended to use this kit toassess distribution for certain classes of compounds. Small moleculeswith a molecular weight below 1 kDa should be evaluated using thecompound distribution kit; molecules larger than 1 kDa are typically nota concern, unless they already proved “sticky” or prone to absorbing oradsorbing to materials used in the experiment during development.Biologics, such as monoclonal antibodies, although less likely to be aconcern, may also be evaluated especially if they are known to be“sticky” or likely to absorb or adsorb to materials used in theexperiment.

In addition to helping assess distribution, the compound distributionkit can also be used to quantitatively account for distribution effects.The quantitative accounting of distribution effects is done usingcalculators that are included in the kit, which can be applied to eachexperiment's results.

The compound distribution kit, in one embodiment, is a kit that allowsusers to approximate the range of possible bio-model exposureconcentrations to a dosed compound over time. The compound distributionkit may be used to determine the absorption of compounds in experimentalor clinical equipment, such as, for example, microfluidic devices. Inone embodiment, the compound distribution kit is intended to be used asa specialized control experiment—the absorption control experiment—priorto an intended study. As such, the contents of the absorption kit may,in one embodiment, mirror the components of an intended study, and themethod of use may be a simplified version of the intended study. Forexample, the compound distribution kit may use the same dosing,perfusion, and sampling design as an intended study or experiment. In anembodiment where these guidelines are followed the compound distributionkit would be able to assess compound distribution (e.g. compound loss orgain) at the same timepoints and under the same experimental conditionsthat are relevant for the intended experiment.

Not only the materials making up microfluidic devices are prone toabsorption. Many materials used in experimental and clinicalapplications absorb small-molecules. For example, infusion tubing usedto administer intravenous fluids and/or drugs may absorb high quantitiesof small-molecule compounds. As another example, the rubber gaskets ofsyringes are also known to absorb small-molecule compounds. Theabsorption of small-molecule compounds into the materials of theinfrastructure used to transport them is especially problematic when thesmall-molecule compounds are, for example, drugs being tested for oreven actively used treat patients. The problem worsens if the patient ispediatric. Pediatric patients receive lower doses or concentrations ofdrugs. If the same fluid transport setup (i.e. the length of infusiontubing) is being used to treat both adult and pediatric patients, thenthe pediatric patients are receiving even less of the drug than theadults. Oftentimes scientists and clinicians are not aware ofsmall-molecule absorption. Physicians and clinical scientists need to beable to both understand if their compound is prone to materialabsorption, and if so, be able to quantify the amount of compound beingabsorbed into their system. In one embodiment, it is recommended thatthe compound distribution kit is used ahead of experiments that involvecompounds that are smaller than 1000 Da. However, if there is anindication that the compound in question suffers from absorption oradsorption in plate-based systems, the compound distribution kit shouldbe used for biologics and small-molecules larger than 1000 Da.

Drug concentration is related to assay results. Generally, whenexperiments are run many compound doses are used. For example, in anexperiment using a drug compound, many drug compound doses may be tried.Following the experiment and relevant assaying, a curve is generatedbased on the system response to the compound. The curve generally has asigmoid. The sigmoid may be upward or downward pointing based on whetherthe compound results in more or less excretion from, for example, cells.As such, the percentage of the compound that is absorbing into thesystem directly affects assay results.

Currently, the primary method of estimating or quantifying compoundabsorption into systems is to do computational modeling of the system,such as with the program COMSOL Multiphysics (COMSOL). However,computational models are oftentimes not an ideal solution. Computationalmodels may not work, as some compounds absorb completely. That is tosay, if systems are exposed to a very low concentration of compound,even if the exposure level can be predicted, it may be too low to be auseful correction. Regardless of the ability to correct data in onlysome situations, computational models also may require a complicatedworkflow.

In order for computational modeling to be functional, absorption ofevery compound introduced into the system should be quantified first inmaterial characterization studies. Material characterization studies canbe very time consuming. Furthermore, fully characterizing systems is notfeasible for large scale experiments or clinical setups is not alwaysfeasible. As well, computational models may not be able to accuratelydeconvolute data in many experiments due to high numbers of variables,including those introduced by the cells. Even with the aid ofcomputational models to account for many of these variables, in thepresence of absorption there is still a decreased overall confidence inresults in in vitro to in vivo extrapolation (IVIVE).

As such, a compound distribution kit is presented herein that may notonly easily inform scientists and clinicians of compound absorption intosystems, but may also, in some embodiments, recommend improvements toreduce absorption. The invention presented herein may be able to savescientists and clinicians valuable time and money over usingcomputational modeling.

Several levels of the compound distribution kit are contemplated. On thesimplest level, the compound distribution kit may offer scientists andclinicians either an affirmative or negative result, alerting themwhether or not the absorption in a system is tolerable. On a slightlymore detailed level, the compound distribution kit may be able to offera range compound absorption. On the most comprehensive level, thecompound distribution kit may be able to fully characterize the system,quantifying the concentration of the compound that is absorbed into thescientist or clinician's system.

In one embodiment, the compound distribution kit characterizes thesystem the scientist or clinician is to use. In a preferred embodiment,however, the compound distribution kit characterizes a simplifiedsystem. A simplified system may be preferable, such that the user doesnot have to spend valuable time or money setting up what may be a verycostly experimental or clinical system. As an example, a holding tankmay be used in the place of a human body if testing the fluidicinfrastructure of a dialysis machine. As another example, a microfluidicdevice with a pore-less membrane separating two channels may be usedinstead of a microfluidic device comprising cultured cells overlaying aporous membrane. If in this example the scientists aim to find if acompound is toxic to the cells within the microfluidic device, they mayuse the same media, take samples at the same time points, etc. In somecases, only portions of a clinical or experimental setup would need tobe understood, and therefore the other components of that system couldbe simplified for use with the compound distribution kit. In oneembodiment, sampling taken for assays may be replaced with samplingtaken for compound concentration analysis.

In one embodiment, a user would design their ideal protocol orexperiment and then modify it accordingly for use with the compounddistribution kit. In one embodiment, the user may use the results of thecompound distribution kit to decide whether the experiment isworthwhile. In one embodiment, the results of the compound distributionkit to modify the experiment, such as to change fluid flow rate, uselower volumes of absorbing material, etc. In one embodiment, the resultsof the compound distribution kit may be used to influence error bars onthe results of the actual protocol. For example, the percent of acompound absorbed may be calculated from the compound distribution kitand those percentages may contribute to error bars following an actualexperiment using the unmodified protocol.

As previously stated, compound concentration is directly related toassay results. Assay results are used to generate half maximalinhibitory concentration calculations IC₅₀. In one embodiment, theresults of the compound distribution kit may be used to influence theIC₅₀ following the completion of an actual protocol. The IC₅₀ is ameasure of the potency of a compound in inhibiting a specific biologicalor biochemical function. In other words, the IC₅₀ is a quantitativemeasure of how much of a compound is needed to inhibit a biologicalprocess. The IC₅₀ represents the concentration of a compound that isneeded for 50% inhibition or maximum effect in vitro, such as in amicrofluidic device. For example, if the intended study shows an IC₅₀ ata dosing concentration of 1 μM, the results of the compound distributionkit may indicate that the actual IC₅₀ is at an exposure concentration inthe range of 0.6 μM to 1 μM.

For example, for a particular protocol using cells within a microfluidicdevice the IC₅₀ at the 24-hour point may be 1 μM. In a worst case all50% of the loss of the compound comes upstream of a microfluidic device.In that worst-case cells within that microfluidic device would only haveseen 0.5 μM of the compound at the IC₅₀. Knowing that the 50% compoundloss happened before the compound entered the microfluidic device wouldaid in adjusting the IC₅₀ graph. In a best-case all of the absorptionhappens downstream of a microfluidic device. In that best-case cellswithin that microfluidic device would have seen 100% of the compounddose. Knowing that all of the absorption happens downstream of themicrofluidic device would mean that the IC₅₀ graph would not need to beadjusted. In another case, the compound is absorbed into themicrofluidic device itself. The case of the compound absorbing into themicrofluidic device itself would lead to the IC₅₀ graph being adjustedsomewhere in between the before mentioned worst-case and best-case.Error bars may also be added to the IC₅₀ graph using the results of thecompound distribution kit. Likewise, the results of the compounddistribution kit may be used to adjust or add error bars to the graphsof assays, such as albumin, lactate dehydrogenase, etc.

In one embodiment, a workflow or method of use for an compounddistribution kit may comprise the steps of (1) prepare experimentalsetup, (2) prepare dosing solutions, (3) dose experimental setup, (4)collect effluent at one or more time points, (5) determine effluentsample concentration, and (6) assess absorption of dosing solution intothe materials making up the experimental setup.

The compound distribution kit may be used with any of the microfluidicdevices presented herein. The compound distribution kit may be used todetermine compound absorption into systems or experiments comprising thehigh-absorbing, gas-permeable microfluidic device, the low-absorbing,gas-impermeable microfluidic device, and the low-absorbing,gas-permeable microfluidic device. The compound distribution kit mayalso be used to determine compound absorption into low-absorbing andhigh-absorbing perfusion manifold assemblies. While the compounddistribution kit may be used by any scientist interested in absorptionof a compound into any system, embodiments particular to themicrofluidic devices and perfusion manifold assemblies discussed hereinwill be presented.

The compound distribution kit for microfluidic device use may comprisephysical and/or digital components. In one embodiment, the physicalcomponent of the compound distribution kit comprises one or moremicrofluidic devices, one or more perfusion manifold assemblies, one ormore filters (such as Millapore brand Steriflip® filters), and a quickstart guide. In one embodiment, the user of the compound distributionkit may also need any of the following: a culture module, a gas mixer,an incubator, a biosafety cabinet, a liquid chromatography-massspectrometer (LCMS), ethanol (such as 70% ethanol), at least one 150 mmPetri dish, at least one 50 mL conical tubes, at least 10 Eppendorf®tubes, pipette tips, a pipette aid or gun, an aspirator, aspirator tips,media, wipes, and dimethyl sulfoxide (DMS)). In one embodiment, saidmicrofluidic device comprises: a) a solid substrate comprising a singlemicrofluidic channel, and b) a non-porous membrane separating saidsingle microfluidic channel into a first chamber and a second chamber. Aquick start guide may be instructions for a user, such that if followedthe user may be able to easily use the compound distribution kit.Filters may be used to equilibrate fluids used in the compounddistribution kit. In an exemplary embodiment, the digital component ofthe compound distribution kit comprises a calculator and a library ofdigital protocols on a community portal. The library of digitalprotocols may comprise one or more protocols.

It is not intended that the present invention be limited by media orstock solution type. The non-dosed media or stock solution may bedimethylsulfoxide (DMSO), water, Eagle's minimal essential medium(EMEM), Dulbecco's modified Eagle's medium (DMEM), etc. Any solvent orcell culture media is imagined. In one embodiment, the microfluidicdevices are washed with 200 μL per channel. In one embodiment, saidperfusion manifold assemblies are primed with 3 mL of media in the inletreservoirs and 200 μL of media in the outlet reservoirs.

In the embodiment in which perfusion manifold assemblies are fluidicallyconnected to at least one culture module, it is recommended a regulationcycle is run immediately before starting an experiment in order todecrease the volume of bubbles in the system. A regulate cycle or bubbleremoval cycle is encompassed in U.S. patent application Ser. No.15/647,727 and is referenced herein in its entirety.

Samples may be taken at any time point the user desires. In an exemplarytimepoint, samples are taken at six time points, including a sampletaken at the beginning of the experiment from each of the perfusionmanifold assembly inlet and outlet reservoirs. For example, if sixsamples are taken from a perfusion manifold assembly comprising twoinlet reservoirs and two outlet reservoirs, there would be a total of 24samples taken for that perfusion manifold assembly. In an exemplaryembodiment, at least three perfusion manifold assemblies are used inorder to achieve better experimental results. For example, if sixsamples are taken from each of three perfusion manifold assemblies eachcomprising two inlet reservoirs and two outlet reservoirs, there wouldbe a total of 72 samples taken for that experiment. When preparing adosing solution portions of both the stock (or blank) solution should beset aside for later solution analysis. In a preferred embodiment, 200 μLof stock and dosing solutions are set aside for later analysis.

A calibration curve, also known as a standard curve, is a general methodfor determining the concentration of a substance in a sample bycomparing the unknown to a set of standard samples of knownconcentration, such as dilutions of 1:10, 1:100, 1:1000, as well asnon-dosed and fully dosed samples. In an exemplary embodiment, samplesolutions are prepared for a five-point standard curve. In an exemplaryembodiment, sample solutions include undiluted dosing solution, a 1:10dosing solution to sample solution dilution, a 1:100 dosing solution tosample solution dilution, a 1:1000 dosing solution to sample solutiondilution, and stock solution. In one embodiment, there are multiple ofeach sample solution in order to decrease experimental error. In oneembodiment, a standard curve is created for each channel of amicrofluidic device. In one embodiment, each perfusion manifold assemblyhas one inlet and one outlet reservoir corresponding to each channel ofa microfluidic device. For example, if a five-point calibration is donefor a single two-channel microfluidic device and corresponding perfusionmanifold assembly with one inlet and one outlet reservoir for eachmicrofluidic device channel, and there are two replicates of eachsample, then 20 total samples would be needed to complete thecalibration curve.

The compound distribution kit may require high numbers of samples,between samples needed for standard curves and samples needed for systemabsorption analysis. For example, in an experiment using threetwo-channel microfluidic devices and three perfusion manifoldassemblies, each with one inlet reservoir and one outlet reservoir, upto 92 samples would be necessary when doing a five-point, two replicatecalibration and taking six timepoint samples at each of the two inletreservoirs and two outlet reservoirs per perfusion manifold assembly. Ifsamples are not used immediately, they should be kept in cold storage,such as a freezer.

Running (or flushing) a culture module at a high flow rate duringexperimentation may be used to prime perfusion manifold assemblies. Inone embodiment, a culture module is run at 600 μL/hr for five minutes tocompletely flush or prime one or more perfusion manifold assembly.

Sample compound concentration quantification may be done in any methodknown in the art. In one embodiment, sample compound concentrations maybe quantified using spectrometry, chromatography, or other separationtechniques. Spectrometry may include mass spectrometry (MS), liquidchromatography-mass spectrometry (LCMS), etc. Chromatography may includehigh performance liquid chromatography (HPLC), thin-layer chromatography(TLC), gas chromatography (GC), counter-current chromatography (CCC),ion chromatography, paper chromatography, etc. Other separationtechniques include centrifugation, electrophoresis, liquid-liquidextraction, solid phase extraction, crystallization, distillation, fieldflow fractionation, drying, decantation, etc.

The calculator may alternatively be known as the absorption calculator.In one embodiment, the calculator outputs the final results of thecompound distribution kit. In one embodiment, the calculator outputsresult that may be able to guide users toward higher accuracy in theirexperiments. The calculator may be used to analytically assess compoundabsorption by comparing the concentration of compound in a plurality ofsaid calibration solutions to the concentration of a compound in one ormore sample solutions. The calculator may be a calculation program orsoftware, such as a Microsoft Excel calculator, a MATLAB calculator,etc. The calculator may also be a script of code, such as Python, C,C++, Java, etc. For example, LCMS results and dosing method may beentered into a “user input” section of a calculator to generate acompound distribution result. In one embodiment, the calculator outputsa graph. In one embodiment, recovered (effluent) concentrations areplotted against time for both top and bottom channels. A recoveredconcentration close to 1 at any given time point, may mean that littlecompound was absorbed by the system. The curves may rise with time asthe gradients that drive absorption and adsorption processes diminish.The range of potential cellular exposure concentrations may be plottedfor each collection time period for one or more channels. For example,if all compound loss occurred upstream of the cells, the cells wouldexperience a lower compound concentration than if the compound lossoccurred entirely downstream of the cells (in which case the cells wouldexperience the full dosing concentration). If a compound is minimallyabsorbed, a user may observe a tighter range near the top of the graph,which means that the cells are expected to be exposed to most of thedosed compound. The calculator may also export a table of results, suchas indicating range of cellular exposure in one or more channels orfraction of dosing values.

Based on the calculator results, a user can choose to proceed with, say,a drug study as is, make a modification to the study (such as change theflowrate or dosing time), or drop the study all together. Based on theresults of the of the compound distribution kit a user might choose tomodify any number of drug study experimental conditions eitherindividually or in combination, depending on the desired impact. Forexample, the user might adjust flowrate in order to create moreconsistent compound exposure concentrations along the length of themicrofluidic device or over time. Indeed, increasing flow rate wouldminimize the time the media, which contains compound, is exposed to theabsorbing material, which in turn minimizes loss of the compound alongthe length of the microfluidic device and creates a more uniformexposure of the cells to the compound. A user might also choose toincrease dosing concentration for a highly absorbing compound, eitheralone or in concert with increasing flow rates. Similarly, a user maydecide to throw out the results from early time points, since this iswhen the extent of compound loss due to absorption is at its highestand, therefore, its effects most impactful. Thus, at later time points,after the system has possibly started to saturate with compound,depending on the absorption characteristics of the particular compoundof interest, and the concentration of the dosed compound begins to rise,the data coming from the system will be more reliable, accurate, andconsistent. For example, for a ±18% uncertainty in a 6 to 24-hour timeperiod may be acceptable for some studies but not others. As a note, ifa user is proceeding with a biological study, the user should measureeffluent compound concentration as well. The calculator is envisioned tooutput varying levels of absorption data depending on the user. At thebroadest end, the calculator would output whether or not the level ofabsorption in a study is allowable, i.e. whether the level of absorptionwas high enough that it would negatively impact the experimental data.Allowable levels of absorption would not be significant in the study.The broad end of calculator output may be considered a go/no-go decisionmaker for experiments for compounds based on results of a simple orsmall-scale experiment. At a slightly more detailed level, thecalculator could report confidence intervals for exposureconcentrations. At an even more detailed level, the calculator wouldoutput quantified absorption levels at the different timepoints, suchthat the user would be able to visualize the absorption of the compoundinto the system for the duration of the experiment. In anotherembodiment, the calculator would tell the user the potential compoundexposure concentration at different time points. Results from absorptiontests, without biologics for example, may be used to put error bars, orconfidence intervals, on exposure concentrations in actual drug studies,comprising biologicals for example. Exposure concentration confidenceintervals decrease with experiment duration, with lower confidence atlater time points. The calculator may output charts for the user to see.Examples of charts include minimal absorption charts, nearly completeabsorption charts, outlet concentration of compound charts, cellularexposure range ranges, dose-response confidence interval charts, etc.For example, the user would enter system input and output concentrationsof a compound and the calculator would then output approximate ranges ofcell exposure to a drug.

The calculator may also output experimental suggestions to lowerabsorbency. In one embodiment, the calculator outputs modifiedexperimental protocols to minimize absorption, such as increased flowrate or waiting until later time points to sample, such as when thesystem has reached steady state.

Again, the digital component of the compound distribution kit maycomprise one or more protocols for the user. In one embodiment, thedigital component of the compound distribution kit comprises protocolsfor running drug studies on a culture module, that defines dosingsolution preparation processes, sampling directions, suggested timepoints for experimental duration. For example, for an experiment of lessthan three hours it may be suggested to sample the system at 0.5, 1,1.5, 2, and 3 hours. For example, for an experiment of six hours it maybe suggested to sample the system at 0.5, 1, 2, 4, and 6 hours. Forexample, for an experiment of 12 hours it may be suggested to sample thesystem at 1, 3, 6, 9, and 12 hours. For example, for an experiment of 24hours it may be suggested to sample the system at 1, 3, 6, 12, and 24hours. For example, for an experiment of 48 hours it may be suggested tosample the system at 1, 3, 6, 24, and 48 hours. For example, for anexperiment of more than 72 hours it may be suggested to sample thesystem at 1, 6, 24, 48, and 72 hours. The digital component of thecompound distribution kit may also comprise a catalog of frequentlyasked questions.

An exemplary method may follow the method of an intended experiment orstudy. As an example, the compound distribution kit may be used withmicrofluidic devices. An exemplary method for use with microfluidicdevices may follow an intended study method with the following changes:no coatings, no cell seeding, and reduced number of compound doses. Inone embodiment, there may not be a need to perform coatings on themicrofluidic device that are related to biological aspects of theexperiment. Some coatings, such as those that change the chemistry ofthe microfluidic device material makeup, may need to be done, especiallyif they may affect compound absorbency. It one embodiment, coatings aredone with the compound distribution kit at the discretion of the user.In one embodiment, cell seeding is not needed. In one embodiment, if theintended-study protocol compares several concentrations of a testcompound, the absorption control experiment need only be run for one ofthese concentrations. In one embodiment, it is recommended to use thehighest compound concentration planned for the intended study, in orderto maximize quantification (e.g. LCMS) sensitivity. However, a lowerconcentration of test compound may be selected if there are concernsabout the compound's solubility limits or it crashing out of solution atthe higher concentration. Crashing out of solution is when theconcentration of a solute in a solution reaches a point where the soluteprecipitates. In one embodiment, the compound is dosed for the sameduration, at the same flow rate, in the same microfluidic channel(s) asper the intended protocol, experiment or study. That is, if the intendedstudy specifies dosing only in a first channel, with no compound in asecond channel, then the same method would be applied during the use ofthe compound distribution kit. In one embodiment, media and solutionshould be the same during use of the compound distribution kit as in theintended study. In one embodiment, the same media or solution, as wellas any additives or supplements, as medium composition and additives caninteract with test compounds (e.g. protein binding). In one embodiment,media or solutions should also be equilibrated in the same manner, suchas degassing and preheating. In one embodiment, effluent samples takenduring use of the compound distribution kit should be collected at thesame time as the intended study. Compound distribution can be a highlydynamic process, and as such matching time points may be able to ensurethat the results of the compound distribution kit correspond closely tothe intended study. In an embodiment where perfusion manifold assembliesare being used, samples may also be taken from each of the inputreservoirs once per input media exchanger, at the same time as effluentsamples are collected right before exchanging media, aiding in ensuringthat no compound is disappearing from the system through an unexpectedmeans, such as compound crashing or photodegradation. In an exemplaryembodiment, it is recommended to generate standard curves for LCMSanalysis. In one embodiment, a 5-point standard curve in triplicateusing a volume of at least 50 μL per sample may be used. Serialdilutions of media used in a first channel and a second channel may beprepared. LCMS may be used to analyze inlet, effluent, blank media andstandard curve samples. The results may, in one embodiment, be put intoa compound distribution or absorption calculator.

In one embodiment, it may be possible to include the modificationsadditively to the first experimental protocol, so that the results ofthe absorption experiment are collected during the intended experiment.This alternative embodiment is still quite useful: it still allowscorrecting the results of the intended study using the addedmeasurements of compound concentration. It may be considered moreefficient to run one experiment instead of two.

An exemplary method of use for the compound distribution kit follows:

1. Media Gas Equilibration

-   -   1. Warm first channel medium and second channel medium in 50 mL        conical tubes at 37° C. in a water or bead bath for at least 1        hour        -   a. Prepare at least 4 mL of each media type to per            microfluidic device—it is recommended to test at least 3            microfluidic devices per compound        -   b. Media should be prepared in the same way as the media            used when dosing cells with compound, matching all media            components/supplements, with the exception of the compound            to be tested at this stage    -   2. Transfer conical tubes to the biosafety cabinet (BSC) and        immediately Steriflip medium:        -   a. Connect the 0.45 μm Steriflip unit to the conical tube            and apply vacuum to assembled unit for 10 seconds prior to            inverting        -   b. Invert the assembled Steriflip and ensure that medium            passes through the filter in a continuous stream        -   c. It should take approximately 2 seconds for each 10 mL of            medium to pass through the filter—if it takes longer, stop            and see troubleshooting protocol as medium will not be            equilibrated properly    -   3. Leave the filtered medium under vacuum for 5 minutes    -   4. Remove conical tube with medium from Steriflip unit while        still under vacuum and then turn off pump. Replace the lid        inside the BSC, and immediately place in an incubator or bath to        maintain temperature    -   5. Store this media with cap slightly loose in the incubator        prior to use

2. Microfluidic Device Washing

-   -   1. Unpackage gamma irradiated microfluidic devices in the BSC        and place in a 150 mm culture dish    -   2. Wash each channel with 200 μL of equilibrated media        -   a. Place the pipette tip perpendicular to channel Inlet        -   b. Ensure tip is snug in port and introduce media into top            and bottom channel        -   c. Aspirate outflow liquid from the outlet of the            microfluidic device    -   3. Aspirate and discard any excess media from the surface of the        microfluidic device, but keep channels filled with media    -   4. If bubbles are observed anywhere in the microfluidic device        channels or ports, aspirate each microfluidic device port to        remove media from channels, then reintroduce media.    -   5. Place small equilibrated medium droplets on each inlet and        outlet    -   6. Cover the culture dish and place in the incubator until Pods        are primed

3. Perfusion Manifold Assembly Priming

-   -   1. Sanitize the exterior of perfusion manifold assembly        packaging with 70% ethanol and transfer perfusion manifold        assemblies into the BSC    -   2. Retrieve trays from the culture module and sanitize with        ethanol before transferring into the BSC        -   a. Orient the trays with the handle to the user's left            inside the BSC    -   3. Open perfusion manifold assembly package in the BSC, and        place the perfusion manifold assemblies into the trays    -   4. Add 3 mL of equilibrated medium to the appropriate inlet        reservoir    -   5. Add 300 μL of equilibrated medium to the appropriate outlet        reservoir, directly over each outlet via    -   6. Prime perfusion manifold assemblies in the culture module        -   a. Use the rotary dial to highlight a priming cycle        -   b. Press the dial to select a prime cycle        -   c. Rotate the dial to a start option and press the dial            again to begin the cycle        -   d. Close the incubator door and wait 1 minute for the cycle            to complete        -   e. The status bar will read ‘Ready’, confirming the cycle is            finished    -   7. Transfer trays to the BSC    -   8. Inspect the underside of each perfusion manifold        assembly—observe droplets have formed on all four ports        -   a. If any perfusion manifold assembly does not show            droplets, re-run a prime cycle on those perfusion manifold            assemblies    -   9. Set perfusion manifold assemblies aside and retrieve        microfluidic devices from the incubator        4. Microfluidic Device to Culture module and Regulate    -   1. Hold perfusion manifold assembly with non-dominant hand    -   2. With microfluidic device in dominant hand, slide the arms of        microfluidic device carrier into the tracks on the underside of        the perfusion manifold assembly until the microfluidic device        Carrier is fully seated in the Perfusion manifold assembly    -   3. Place thumb on the carrier tab and gently depress tab in and        up to engage the tab with the perfusion manifold assembly    -   4. Aspirate any excess medium from perfusion manifold assembly        window    -   5. Place the perfusion manifold assembly with microfluidic        device into the tray, with the reservoirs along the back wall    -   6. Repeat for each perfusion manifold assembly and microfluidic        device Carrier and transfer loaded Tray to Zoë    -   7. Select flow rate settings on Zoë        -   a. Flow Rate: 30 μL/hr for the top and bottom channel    -   8. Run a regulate cycle to reduce bubble formation        -   a. The cycle will take 2 hours to complete, after which the            culture module switches to the set flowrate

5. Second Regulate Cycle

-   -   1. The following morning of running the regulate cycle, pause        the culture module by pressing the silver activation button        located over the bays    -   2. Slide tray out and transfer to the BSC    -   3. Remove perfusion manifold assembly lids and using a 200 μl        pipette, perform a via wash on each inlet and outlet perfusion        manifold assembly reservoir:        -   a. Using media within the perfusion manifold assembly            reservoir, pipette 200 μL of media directly over the top of            the via to dislodge any bubbles that may be present        -   b. Repeat this wash step for each of the four perfusion            manifold assembly reservoirs    -   4. Replace perfusion manifold assembly lids and return the Trays        to the culture    -   5. Run the regulate cycle again    -   6. Dosing is ready to commence following completion of the        second regulate cycle

6. Preparation of Dosing Solution

Note 1: Use the same media with supplements that will be used forrunning the microfluidic device study for the system absorption test.

-   -   1. Refer to the Calculator for Study Design and Data Handling        for this portion of the protocol        -   a. In a “USER INPUTS” tab, add details of the planned dosing            experiment in the designated space (flowrate, duration of            study, units of concentration, channel to be dosed with            compound).        -   b. Use the dropdown menu to select the channel to be dosed            with compound            -   i. First            -   ii. Second            -   iii. First and second    -   2. Prepare the stock solution of compound by dissolving in        vehicle of choice, based on the dosing volume indicated in the        calculator    -   3. Dilute stock solution in the appropriate gas-equilibrated        media

7. Dosing and Sample Collection

-   -   1. Pause the culture module    -   2. Take trays to the BSC    -   3. Aspirate the media out of the inlets and outlets making sure        to avoid bringing the aspirator tip too close to the vias (there        will be a small amount of media remaining near the via and this        is acceptable).    -   4. Refer to the calculator and add total media volume needed to        run the study to completion into the top and bottom inlet        reservoirs of the perfusion manifold assembly.    -   5. Sample 50 μL from the top and bottom inlet reservoir of each        perfusion manifold assembly to capture the t=0 dosing media        concentration.    -   6. Reserve 200 μL dosing media from the conical as well as        “blank” media with no compound for standard curve preparation        -   a. Store these samples according to user standard practices    -   7. Return the tray into the culture module and prime the system        with dosing media by setting the flow rate to 600 μL/hr and run        for 5 minutes. This replaces the media in the microfluidic        device with dosing solution.    -   8. Pause the culture module and transport trays to the BSC    -   9. Completely aspirate the effluent collected in the outlet        reservoir so as not to dilute the compound effluent collected in        the later timepoints.    -   10. Return the tray to the culture module and set the flow rate        as directed in the study. Begin timing for sample collection        once flow is initiated on the culture module    -   11. Use the remaining dosing solution to prepare serial        dilutions as samples for a standard calibration curve        -   a. It is recommend to generate samples for a 5-point            standard curve in a triplicate of volume 50 μL using serial            dilutions of the top and bottom media using the following            ratios (Dosing solution:Media)        -   i. Undiluted media with compound        -   ii. 1:10 dilution        -   iii. 1:100 dilution        -   iv. 1:1000 dilution        -   v. Blank (media without compound)        -   b. Store these samples according to user standard practices    -   12. Sample 50 μL from inlet and outlet reservoir at the        remaining timepoints until the conclusion of the study        -   a. With the exception of the first and last timepoints,            subsequent sample times do not require sampling the inlet            reservoirs        -   b. Handle and process samples per user standard practices        -   c. Aspirate outlets completely before returning perfusion            manifold assemblies to the culture module            NOTE: In case the volume collected is less than 50 μL,            record the volume collected.    -   13. Send samples to LCMS upon completion of the dosing        experiment.

8. Data Analysis

-   -   14. Upon sample analysis by LCMS, enter the concentration data        into the “User Inputs” sheet in columns D, E, F of the        calculator into the appropriate cells    -   15. View the results in the appropriate tab marked.        -   a. Recovered concentrations are plotted with time for both            top and bottom channels.    -   b. The range of potential cellular exposure concentrations are        plotted for each collection time period and both channels.    -   c. Tables indicate the range (max and min) cellular exposure        concentrations in both channels with time as well as the        exposure expressed as a fraction of the dosing concentration.

One embodiment of the present invention is a method of analyzingcompound distribution in a system, comprising: a) providing a system anda first experimental protocol for use with said system, said firstexperimental protocol comprising introducing a compound into said systemand taking actions at one or more timepoints; b) modifying said firstexperimental protocol to generate a first modified experimentalprotocol; c) measuring compound concentration at one or more of saidtimepoints from said first experimental protocol; d) performing saidfirst modified experimental protocol; and e) using said measurement ofconcentration of said compound to analyze compound distribution acrosssaid system. In one embodiment, the method further comprises the step off) performing said first experimental protocol. In one embodiment, saidsystem comprises one or more microfluidic devices. In one embodiment,said system comprises infusion tubing. In one embodiment, said systemcomprises syringes. In one embodiment, said system comprises one or morebiological elements and said first experimental protocol is modified toexclude at least one of said one or more biological elements. In oneembodiment, said first experimental protocol comprises compound testingon said biological elements. In one embodiment, said first experimentalprotocol comprises cells and said first modified experimental protocoldoes not comprise cells. In one embodiment, said system comprisescoatings and said first experimental protocol is modified by excludingcoatings. In one embodiment, said first modified experimental protocoldoes not comprise taking actions at one or more timepoints of said firstexperimental protocol. In one embodiment, said performing a measurementof the concentration replaces said taking actions at one or moretimepoints. In one embodiment, said first modified experimental protocolis modified in that only a subset of input compound concentrations areincluded in said modified experimental protocol as compared to saidfirst experimental protocol. In one embodiment, said first modifiedexperimental protocol in that porous elements are excluded as comparedto said first experimental protocol. In one embodiment, said systemincludes a first microfluidic device comprising a first membrane withpores. In one embodiment, said system is replaced with a second systemin said modified experimental protocol, said second system including asecond microfluidic device not comprising a membrane without pores in atleast one region in which said first membrane comprises pores. In oneembodiment, said first experimental protocol comprises flowing fluid insaid system. In one embodiment, said system comprises an input portconfigured to permit fluid input to the system. In one embodiment, thesystem comprises an output port configured to permit fluid output fromthe system. In one embodiment, said first experimental protocolcomprises flowing into said input port. In one embodiment, said firstexperimental protocol comprises collecting a first sample from saidoutput port. In one embodiment, said measuring of the concentration ofsaid compound comprises collecting a sample from said output port andquantifying said concentration of said compound in said sample. In oneembodiment, said first modified experimental protocol further quantifiesthe percentage of said compound that is absorbed into said system. Inone embodiment, the method further comprises introducing fluid flow tosaid system. In one embodiment, said taking actions comprises samplingeffluent. In one embodiment, said first experimental protocol furthercomprises assaying said effluent to achieve an apparent metabolitevalue. In one embodiment, the method further comprises using saidmeasurement of concentration of said compound to correct said apparentmetabolite value. In one embodiment, the method further comprises usingsaid measurement of concentration of said compound to determinevariability of said apparent metabolite value. In one embodiment, themethod further comprises using said measurement of concentration todetermine whether to perform said first experimental protocol. In oneembodiment, the method further comprises (i) using said measurement ofconcentration of said compound to generate a second modifiedexperimental protocol; and (ii) performing said second modifiedexperimental protocol. In one embodiment, said first experimentalprotocol comprises living cells.

The present invention contemplates, in one embodiment, a method ofdetermining compound distribution in a system, comprising: a) providinga first system and a first experimental protocol for a said firstsystem, said first system comprising: i) first fluidic channel; ii) asecond fluidic channel; and iii) a first membrane disposed between saidfirst fluidic channel and said second fluidic channel, said firstmembrane comprising pores; wherein said first experimental protocolcomprises introducing a compound into said first system and takingactions at one or more timepoints; b) modifying said first experimentalprotocol to generate a first modified experimental protocol, bysubstituting said first membrane with a second membrane, said secondmembrane lacking pores; c) performing said modified experimentalprotocol; d) performing a measurement of the concentration of saidcompound at one or more of said timepoints of said first experimentalprotocol; and e) comparing said measurement of concentration of saidcompound to the concentration of said compound to determine compounddistribution in said system. In one embodiment, said taking actionscomprises sampling an effluent. In one embodiment, the method furthercomprises said effluent. In one embodiment, said experimental protocolcomprises one or more biological elements. In one embodiment, said firstexperimental protocol is modified by excluding at least one of saidbiological elements. In one embodiment, said biological elementscomprise cells. In one embodiment, said biological elements comprisebiological coatings. In one embodiment, said modified experimentalprotocol determines the compound absorption into said system bycalculating the percentage of said compound that is absorbed into thesetup of said experimental protocol. In one embodiment, saidexperimental protocol comprises contacting said one or more biologicalelements with said compound.

The present invention contemplates, in one embodiment, a method ofdetermining compound distribution in a system, comprising: a) providinga system and an experimental protocol for said system comprising one ormore biological elements; wherein said one or more biological elementsare contacted by a compound; b) modifying said experimental protocol byexcluding at least one of said one or more biological elements; c)performing said modified experimental protocol; and d) determining thedistribution of said compound in said system using by measuring theconcentration of said compound in said system. In one embodiment, saidexperimental protocol comprises introducing fluid flow into said system.In one embodiment, the method further comprises collecting effluent. Inone embodiment, said experimental protocol comprises assaying saideffluent. In one embodiment, said biological elements comprise cells. Inone embodiment, said biological elements comprise biological coatings.In one embodiment, said system comprises one or more microfluidicdevices. In one embodiment, said distribution of said compound is usedto calculate error bars for results from said experimental protocol. Inone embodiment, percent distribution of said compound is used tocalculate half maximal inhibitory concentration (IC₅₀) for saidexperimental protocol

The present invention contemplates, in one embodiment, a method ofassessing compound distribution in a system, comprising: a) providing asystem and a first experimental protocol for said system, said firstexperimental protocol comprising introducing a compound into saidsystem; b) modifying said first experimental protocol to generate amodified experimental protocol, said modified experimental protocolcomprising: i) introducing said compound using a first concentration;and ii) performing a first measurement of the concentration of saidcompound; c) performing said modified experimental protocol; d)comparing said measurement of the concentration of said compound to athreshold; e) performing said first experimental protocol if saidmeasurement of concentration surpasses said threshold. In oneembodiment, said first experimental protocol further comprisesintroducing fluid flow into said system. In one embodiment, the firstexperimental protocol further comprises collecting effluent at one ormore time points. In one embodiment, said first experimental protocolcomprises assaying said effluent. In one embodiment, said biologicalelements comprise cells. In one embodiment, said biological elementscomprise biological coatings. In one embodiment, said system comprisesone or more microfluidic devices. In one embodiment, said firstmeasurement is performed at least one of said one or more time points ofsaid first experimental protocol. In one embodiment, said measurement ofthe concentration of said compound to a threshold are compared bydividing said first measurement by said first concentration to obtain afirst ratio. In one embodiment, the said threshold is a first ratiovalue above one of 10%, 20%, 33%, 50%, 66%, and 75%. In one embodiment,said modified experimental protocol further comprises measuring an inputcompound concentration, and wherein the said first measurement isdivided by the measured said input concentration to obtain a measuredratio.

One embodiment of the present invention is a method of assessingcompound distribution, comprising: a) introducing a flow to a fluidiccircuit, said flow comprising an initial concentration of a compound; b)collecting one or more effluent samples from said fluidic circuit; c)determining the concentration of said compound in said one or moreeffluent samples so as to generate measured concentrations; and d)comparing said measured concentrations with the initial concentration ofsaid compound, thereby assessing compound absorption in said fluidiccircuit. In one embodiment, said fluidic circuit comprises one or moremicrofluidic devices. In one embodiment, said one or more microfluidicdevices comprise at least one inlet and/or one outlet. In oneembodiment, the fluidic circuit further comprises one or more perfusionmanifold assemblies in fluidic communication with said one or moremicrofluidic devices. In one embodiment, said fluidic circuit comprisesinfusion tubing. In one embodiment, said fluidic circuit comprises oneor more syringes. In one embodiment, said fluidic circuit comprises apolymer that absorbs small-molecules. In one embodiment, saidconcentrations of said compound in one or more effluent samples aredetermined using chromatography and/or spectrometry. In one embodiment,said concentrations of said compound in one or more effluent samples aredetermined using liquid chromatography-mass spectrometry (LCMS). In oneembodiment, said compound is a small-molecule compound. In oneembodiment, said compound is a drug.

In one embodiment, a method of assessing compound absorption into asystem is contemplated, comprising: a) defining an experimental protocolfor a system; b) modifying said experimental protocol to excludebiological elements; c) performing said modified experimental protocolto assess compound absorption into said system. A method of assessingcompound absorption into a system is contemplated, comprising: a)defining an experimental protocol for a system comprising one or moremicrofluidic devices comprising one or more porous elements; b)modifying said experimental protocol to substitute said one or moremicrofluidic devices comprising one or more porous element with one ormore microfluidic devices comprising non-porous elements; c) performingsaid modified experimental protocol to assess compound absorption intosaid system. A method of assessing compound absorption into a system iscontemplated, comprising: a) defining an experimental protocol for asystem; b) modifying said experimental protocol by excluding biologicalelements; c) performing said modified experimental protocol to assessthe percent absorption of a compound into said system over time, whereinsaid percent absorption of said compound is used to calculate error barcalculations for said experimental protocol over the duration of theexperiment.

In one embodiment, a method of assessing compound absorption is,contemplated, comprising: a) defining an experimental protocol for asystem comprising the use of a compound; b) modifying said experimentalprotocol to exclude biological elements; c) performing said modifiedexperimental protocol to assess the absorption of said compound intosaid system; and d) performing said experimental protocol if less than50% of said compound is absorbed into said system at a time point ofinterest. In one embodiment, said experimental protocol comprisescollecting effluent. In one embodiment, said experimental protocolcomprises assaying said effluent. In one embodiment, said biologicalelements comprise cells. In one embodiment, said biological elementscomprise biological coatings. In one embodiment, said system comprisesone or more microfluidic devices. In one embodiment, compound absorptioninto said system is less than 40%. In one embodiment, compoundabsorption into said system is less than 30%. In one embodiment,compound absorption into said system is less than 20%. In oneembodiment, compound absorption into said system is less than 10%. Inone embodiment, compound absorption into said system is less than 5%.

In one embodiment, a method of assessing compound distribution in asystem is contemplated, comprising: a) providing an experimental orclinical system, b) defining a first experimental protocol for a system,said first experimental protocol comprising introducing a compound intosaid system and taking actions at one or more timepoints; c) modifyingsaid first experimental protocol to generate a modified experimentalprotocol, said modified experimental protocol comprising performing ameasurement of the concentration of said compound at one or more of saidtimepoints from said first experimental protocol; d) performing saidmodified experimental protocol; and e) using said measurement ofconcentration of said compound to assess compound distribution. In oneembodiment, the method further comprises the step off) performing saidfirst experimental protocol. In one embodiment, said system comprisesone or more microfluidic devices. In one embodiment, said systemcomprises infusion tubing. In one embodiment, system comprises syringes.In one embodiment, said system further comprises pipette tips. In oneembodiment, said system further comprises culture plates. In oneembodiment, said system comprises biological elements and said firstexperimental protocol is modified to exclude biological elements. In oneembodiment, said experimental protocol comprises compound testing onsaid biological elements. In one embodiment, said system comprises cellsand said first experimental protocol is modified by excluding cells. Inone embodiment, said system comprises coatings and said firstexperimental protocol is modified by excluding coatings. In oneembodiment, said experimental protocol is modified by removing saidtaking actions at one or more time points. In one embodiment, saidperforming a measurement of the concentration of said taking actions. Inone embodiment, said experimental protocol is modified by selecting onlya subset of input compound concentrations to include in said modifiedexperimental protocol. In one embodiment, said experimental protocol ismodified by excluding porous elements. In one embodiment, said systemincludes a first microfluidic device comprising a first membrane withpores; and further comprising replacing said system with a secondsystem, said second system including a second microfluidic device notcomprising a membrane without pores in at least one region in which saidfirst membrane comprises pores. In one embodiment, said firstexperimental protocol comprises flowing fluid in said system. In oneembodiment, said system comprises an input port configured to permitfluid input to the system. In one embodiment, the system comprises anoutput port configured to permit fluid output from the system. In oneembodiment, said first experimental protocol comprises flowing into saidinput port. In one embodiment, said first experimental protocolcomprises collecting a first sample from said output port. In oneembodiment, said performing a measurement of the concentration of saidcompound of the said modified experimental protocol comprises collectinga concentration sample from said output port. In one embodiment, saidmodified experimental protocol further assesses the percentage of saidcompound that is absorbed into said system. In one embodiment, saidtaking actions comprises sampling effluent. In one embodiment, saidfirst experimental protocol further comprises assaying said effluent toachieve a result. In one embodiment, said measurement of concentrationof said compound to correct said result. In one embodiment, saidmeasurement of concentration of said compound to determine variabilityof said result. In one embodiment, said measurement of concentration todetermine whether to perform said first experimental protocol. In oneembodiment, the method further comprises (i) using said measurement ofconcentration of said compound to generate a second modifiedexperimental protocol; and (ii) performing said second modifiedexperimental protocol. In one embodiment, said system comprises livingcells.

In one embodiment, a method of assessing compound distribution in asystem is contemplated, comprising: a) defining a first experimentalprotocol for a first system, said first system comprising: i) firstfluidic channel; ii) a second fluidic channel; and iii) a first membraneinterspersed in at least one region between said first fluidic channeland said second fluidic channel, said first membrane comprising pores;wherein said first experimental protocol comprises introducing acompound into said first system and taking actions at one or moretimepoints; b) modifying said first experimental protocol to generate amodified experimental protocol, by substituting said first membrane witha second membrane, said second membrane lacking pores in at least onecorresponding region in which said first membrane comprises pores;wherein said modified experimental protocol comprises performing ameasurement of the concentration of said compound at one or more of saidtime points of said first experimental protocol; c) performing saidmodified experimental protocol; and d) using said measurement ofconcentration of said compound to assess compound absorption. In oneembodiment, said taking actions comprises sampling an effluent. In oneembodiment, the method further comprises assaying said effluent. In oneembodiment, said experimental protocol is modified by excludingbiological elements. In one embodiment, said biological elementscomprise cells. In one embodiment, said biological elements comprisebiological coatings. In one embodiment, said modified experimentalprotocol assesses the compound absorption into said system by assessingthe percentage of said compound that is absorbed into the setup of saidexperimental protocol. In one embodiment, said experimental protocolcomprises compound testing on said biological elements.

In one embodiment, a method of assessing compound distribution in asystem is contemplated, comprising: a) defining an experimental protocolfor a system; b) modifying said experimental protocol by excludingbiological elements; c) performing said modified experimental protocolto assess the percent absorption of a compound into said system, whereinsaid percent absorption of said compound to calculate results for saidexperimental protocol. In one embodiment, said experimental protocolcomprises collecting effluent. In one embodiment, said experimentalprotocol comprises assaying said effluent. In one embodiment, saidbiological elements comprise cells. In one embodiment, said biologicalelements comprise biological coatings. In one embodiment, said systemcomprises one or more microfluidic devices. In one embodiment, saidexperimental protocol comprises a compound. In one embodiment, saidexperimental protocol comprises testing compound on said biologicalelements. In one embodiment, said percent absorption of said compound isused to calculate error bars for said results from said experimentalprotocol. In one embodiment, said percent absorption of said compound isused to calculate half maximal inhibitory concentration (IC₅₀) for saidexperimental protocol

The present invention contemplates, in one embodiment, a method ofassessing compound distribution in a system, comprising: a) defining afirst experimental protocol for a system, said first experimentalprotocol comprising introducing a compound into said system; b)modifying said first experimental protocol to generate a modifiedexperimental protocol, said modified experimental protocol comprising:i) introducing said compound using a first concentration; and ii)performing a first measurement of the concentration of said compound; c)performing said modified experimental protocol; d) comparing saidmeasurement of the concentration of said compound to a threshold; and e)performing first experimental protocol if said measurement ofconcentration surpasses said threshold. In one embodiment, said firstexperimental protocol comprises collecting effluent. In one embodiment,said first experimental protocol comprises assaying said effluent. Inone embodiment, said biological elements comprise cells. In oneembodiment, said biological elements comprise biological coatings. Inone embodiment, said system comprises one or more microfluidic devices.In one embodiment, said first measurement is performed at one or more ofthe same timepoints as in the first experimental protocol. In oneembodiment, said comparing in (d) comprises dividing said firstmeasurement by said first concentration to obtain a first ratio. In oneembodiment, said threshold is a first ratio value above one of 10%, 20%,33%, 50%, 66%, and 75%. In one embodiment, said modified experimentalprotocol further comprises measuring an input compound concentration,and wherein the said first measurement is divided by the measured saidinput concentration to obtain a measured ratio.

In one embodiment, a method of assessing compound absorption iscontemplated, comprising: a) introducing a compound to a fluidiccircuit; b) collecting one or more effluent samples from said fluidiccircuit; c) determining the concentration of said compound in said oneor more effluent samples so as to generate measured concentrations; andd) comparing said measured concentrations with the concentration of saidcompound, thereby assessing compound absorption in said fluidic circuit.In one embodiment, said fluidic circuit comprises one or moremicrofluidic devices. In one embodiment, each of said one or moremicrofluidic devices comprise at least one inlet. In one embodiment,each of said one or more microfluidic devices comprise at least oneoutlet. In one embodiment, said fluidic circuit comprises one or moreperfusion manifold assemblies in fluidic communication with said one ormore microfluidic devices. In one embodiment, said fluidic circuitcomprises infusion tubing. In one embodiment, said fluidic circuitcomprises one or more syringes. In one embodiment, said fluidic circuitcomprises a polymer that absorbs small-molecules. In one embodiment,said concentrations of said compound in one or more effluent samples aredetermined using chromatography and/or spectrometry. In one embodiment,said concentrations of said compound in one or more effluent samples aredetermined using liquid chromatography-mass spectrometry (LCMS). In oneembodiment, said compound is a small-molecule compound. In oneembodiment, said compound is a drug. In one embodiment, said fluidiccircuit further comprises one or more perfusion manifold assemblies. Inone embodiment, said fluidic circuit further comprises at least oneinlet.

In one embodiment, a workflow or method of use for an compounddistribution kit may comprise the steps of (1) prepare microfluidicdevices and perfusion manifold assemblies for use with a culture module,(2) prepare one or more dosing solutions for calibration, (3) dosemicrofluidic devices and perfusion manifold assemblies, (4) collecteffluent at desired time points, (5) quantify effluent compoundconcentration, and (6) assess system absorption of compound. A method ofassessing compound absorption is contemplated, comprising: a) providinga compound, a stock solution and a fluidic circuit comprising a fluidicdevice and at least one inlet and at least one outlet; b) combining saidcompound and said stock solution so as to prepare a dosing solution anda plurality of calibration solutions; c) introducing at least a portionof said dosing solution into said fluidic device at one or more of saidat least one inlet; d) collecting one or more effluent samples from oneor more of said at least one outlet; e) determining the concentration ofsaid compound in said one or more effluent samples so as to generatemeasured concentrations; and f) comparing said measured concentrationswith the concentration of said compound in said plurality of calibrationsolutions, thereby assessing compound absorption in said fluidiccircuit. In one embodiment, said concentrations of said compound in oneor more effluent samples are determined using chromatography and/orspectrometry. In one embodiment, said concentrations of said compound inone or more effluent samples are determined using liquidchromatography-mass spectrometry (LCMS). In one embodiment, saidcompound is a small-molecule compound. In one embodiment, said compoundis a drug. In one embodiment, said calibration solutions comprise afive-point calibration. In one embodiment, said fluidic circuit furthercomprises one or more perfusion manifold assemblies.

In one embodiment, a method of assessing compound absorption into a flowsystem is contemplated comprising: a. providing one or more microfluidicdevices, one or more perfusion manifold assemblies, one or more culturemodules, a compound, and a stock solution; b. preparing one or moredosing solutions and one or more calibration solutions by dosing saidstock solution with said compound; c. priming said one or moremicrofluidic devices and said one or more perfusion manifold assemblieswith said stock solution; d. fluidically connecting said one or moremicrofluidic devices, said one or more perfusion manifold assemblies,and said one or more culture modules as to create a flow system; e.replacing said stock solution with said dosing solution in said one ormore microfluidic devices and said one or more perfusion manifoldassemblies; f. collecting a plurality of effluent solutions at one ormore time points; g. determining the concentration of said compound insaid plurality effluent solutions; and h. comparing the concentration ofsaid compound in said plurality of effluent solutions and said one ormore calibration solutions, thereby assessing compound absorption intosaid flow system. In one embodiment, said one or more microfluidicdevices each comprise at least one inlet and at least one outlet. In oneembodiment, said one or more perfusion manifold assemblies each compriseat least one inlet and at least one outlet. In one embodiment, saidconcentrations of said compound in said plurality of effluent samplesare determined using chromatography and/or spectrometry. In oneembodiment, said concentrations of said compound in said plurality ofeffluent samples are determined using liquid chromatography-massspectrometry (LCMS). In one embodiment, said compound is asmall-molecule compound. In one embodiment, said compound is a drug. Inone embodiment, said one or more calibration solutions comprise afive-point calibration.

In one embodiment, a method of assessing compound absorption into a flowsystem is contemplated comprising: a. providing one or more microfluidicdevices, one or more perfusion manifold assemblies, at least one culturemodule, a compound, and a stock solution; b. preparing said one or moremicrofluidic devices and said one or more perfusion manifold assembliesfor use with said one or more culture modules by: i. priming said one ormore microfluidic devices with said stock solution; and ii. fluidicallyconnecting said one or more microfluidic devices to said one or moreperfusion manifold assemblies, and said one or more perfusion manifoldassemblies to said at least one culture module as to create a flowsystem; c. preparing a dosing solution and one or more calibrationsolutions by dosing said stock solution with said compound; d.introducing said dosing solution into said one or more microfluidicdevices by: i. fluidically disconnecting said one or more perfusionmanifold assemblies from said at least one culture module; ii. replacingsaid stock solution with said dosing solution; and iii. fluidicallyconnecting said one or more perfusion manifold assemblies to said atleast one culture module; e. sampling effluent from said flow system atone or more time points to create one or more effluent samples; d.determining the concentration of said compound in said one or morecalibration solutions, said one or more effluent samples, said dosingsolution, and said stock solution; and e. comparing the concentration ofsaid compound in said one or more calibration solutions, said one ormore effluent samples, said dosing solution, and said stock solution,thereby assessing compound absorption into said flow system.

In another embodiment, a method of assessing compound absorption into aflow system is contemplated comprising: a. providing one or moremicrofluidic devices, one or more perfusion manifold assemblies eachcomprising at least one inlet reservoir and at least one outletreservoir, at least one culture module, a compound, and a stocksolution; b. preparing microfluidic devices and perfusion manifoldassemblies for use with said at least one culture module by: i.degassing said stock solution using a filter; ii. washing said one ormore microfluidic devices with said stock solution; iii. priming one ormore perfusion module assemblies by partially or completely filling saidat least one inlet reservoir and said at least one outlet reservoir withsaid degassed stock solution; and iv. fluidically connecting said one ormore microfluidic devices with said one or more perfusion manifoldassemblies and fluidically connecting said one or more perfusionmanifold assemblies with said at least one culture module as to create aflow system; c. preparing a dosing solution by dosing said stocksolution with said compound; d. introducing said dosing solution intosaid one or more microfluidic devices and said one or more perfusionmanifold assemblies by: i. fluidically disconnecting said one or moreperfusion manifold assemblies from said at least one culture module andremoving any of said stock solution left in said at least one inletreservoir; ii. partially or completely filling said at least one inletreservoir with said dosing solution and setting aside a portion of eachof said dosing solution and said stock solution; iii. fluidicallyconnecting said one or more perfusion manifold assemblies to said atleast one culture module and flushing said at least one culture moduleat a high flow rate for at least five minutes; iv. stopping said atleast one culture module from running, fluidically disconnecting saidone or more perfusion manifold assemblies from said at least one culturemodule, and aspirating the resulting effluent from said at least oneoutlet reservoir; v. fluidically connecting said one or more perfusionmanifold assemblies to said at least one culture module, running said atleast one culture module at a low flow rate for a desired period oftime; and vi. sampling from said at least one inlet reservoir and saidat least one outlet reservoir at planned time points to yield one ormore samples; e. preparing a plurality of calibration solutions for afive-point standard curve, said plurality of calibration solutionscomprising i. at least one sample of undiluted dosing solution, ii. atleast one sample of a 1:10 dilution of said dosing solution to saidstock solution, iii. at least one sample of a 1:100 dilution of saiddosing solution to said stock solution, iv. at least one sample of a1:1000 dilution of said dosing solution to said stock solution, and v.at least one sample of said stock solution, f. determining theconcentration of said compound in said plurality of calibrationsolutions, said one or more samples, said dosing solution, and saidstock solution through using chromatography and/or spectrometry; and g.comparing the concentration of said compound in said one or morecalibration solutions, said one or more samples, said dosing solution,and said stock solution, thereby assessing compound absorption into saidflow system.

In one embodiment, a workflow or method of use for an compounddistribution kit may comprise the steps of (1) prepare one or moremicrofluidic devices and perfusion manifold assemblies for use with aculture module by (a) washing said one or more microfluidic devices witha media and (b) fluidically connecting said one or more microfluidicdevices with at least culture module, (2) prepare one or more dosingsolutions by (a) preparing stock solutions and (b) dosing said stocksolution with a compound, (3) dose microfluidic devices and perfusionmanifold assemblies by (a) fluidically disconnecting said one or moreperfusion manifold assemblies from said at least one culture module, (b)replacing said media with said at least one dosing solution, and (c)fluidically connecting said one or more perfusion manifold assembliesfrom said at least one culture module, (4) quantifying sample compoundconcentration by (a) taking sample solutions from said outlet reservoirsat one or more timepoints (b) preparing a plurality of calibrationsolutions for a calibration curve and (c) analytically quantifying theconcentration of said compound in all of said sample solutions and saidplurality of calibration solutions and (5) analytically assessingcompound absorption by comparing the concentration of said compound insaid plurality of said calibration solutions to the concentration ofsaid compound in said one or more sample solutions.

In one embodiment, a workflow or method of use for an compounddistribution kit may comprise the steps of (1) prepare microfluidicdevices and perfusion manifold assemblies, comprising at least one inletreservoir and at least one outlet reservoir, for use with a culturemodule by (a) degassing media using a filter and (b) washing said one ormore microfluidic devices with said degassed media, and (c) fluidicallyconnecting said one or more microfluidic devices with said culturemodule by (i) priming one or more perfusion module assemblies with saiddegassed media by filling said at least one inlet reservoir and at leastone outlet reservoir with media, (ii) fluidically connecting said one ormore microfluidic devices with said one or more perfusion manifoldassemblies, and (iii) placing said one or more perfusion manifoldassemblies in fluidic communication with at least one culture module,(2) prepare a dosing solution by (a) preparing a stock solution and (b)dosing said stock solution with a compound as to create a dosingsolution, (3) dose microfluidic devices and perfusion manifoldassemblies by (i) fluidically disconnecting said one or more perfusionmanifold assemblies from said at least one culture module and removingany media left in said at least one inlet reservoir, (ii) filling saidat least one inlet reservoir with said dosing solution and setting asidea portion of each of said dosing solution and said stock solution, (iii)fluidically connecting said perfusion manifold assemblies to said atleast one culture module and flushing said at least one culture moduleat a high flow rate for five minutes, (iv) stopping said at least oneculture module from running, removing said one or more perfusionmanifold assemblies from said at least one culture module and aspiratingthe resulting effluent from said at least one outlet reservoir, (v)fluidically connecting said one or more perfusion manifold assemblies tosaid at least one culture module, and (vi) sampling from said inlet andoutlet reservoirs at planned timepoints to yield one or more samplesolutions, (4) quantifying sample compound concentration by (a)preparing a plurality of calibration solutions for a five point standardcurve, said plurality of calibration solutions comprising (i) at leastone sample of undiluted dosing solution, (ii) at least one sample of a1:10 dilution of said dosing solution to said stock solution, (iii) atleast one sample of a 1:100 dilution of said dosing solution to saidstock solution, (iv) at least one sample of a 1:1000 dilution of saiddosing solution to said stock solution, and (v) at least one sample ofsaid stock solution, (b) analytically quantifying the concentration ofsaid compound across said plurality of calibration solutions and saidone or more sample solutions, and (5) analytically assessing compoundabsorption by comparing the concentration of said compound in saidplurality of said calibration solutions to the concentration of saidcompound in said one or more sample solutions using an absorptioncalculator.

The present invention is also related to gas distribution withinmicrofluidic devices. Several methods are contemplated for controllinggas distribution within microfluidic devices.

One embodiment contemplated to control gas is a microfluidic devicecomprising one or more gas-exchange channels to flow a fluid, either agas or liquid, and exchange gas between a gas source and another one ormore channels within a microfluidic device. The gas-control microfluidicdevice allows the gas concentration within a gas-permeable microfluidicdevice to be controllable. A gas, such as oxygen, nitrogen, helium,carbon dioxide, a mixture thereof, a smoke, a vapor, etc., may beintroduced into the gas channels of the microfluidic device. The body ofthe microfluidic device comprises a permeable material, such as PDMS.The gas may transport through the body of the microfluidic device intothe working or cell channels of the microfluidic device. Cell viabilitymay be improved when the cells are cultured in similar environments thatthey experience in vivo. As such, the ability to introduce in vivorelevant gas concentrations to the cells within the microfluidic deviceallows scientists to achieve better experimental results. For example,if an anaerobic environment is desired for the channels, nitrogen may beflowed through the gas channels. For another example, if a highlyoxygenated environment is desired for the channels, oxygen may be flowedthrough the gas channels.

In one embodiment, a microfluidic device comprises a body having aculture channel, a gas-exchange channel, and a gas exchanger betweensaid culture channel and said gas-exchange channel. In one embodiment,said gas-exchange channel comprises a gas. In one embodiment, saidgas-exchange channel comprises a fluid or liquid.

Another embodiment contemplated to control gas is a “halo chip,” amicrofluidic device with the capability of creating a desired gaseousenvironment within the channels of the microfluidic device. The “halochip” or gas control microfluidic device has a gas channel orgas-exchange channel that runs around the perimeter of the working orcell channels of the microfluidic device. In one embodiment, saidgas-exchange channel comprises a gas. In one embodiment, saidgas-exchange channel comprises a fluid.

In one embodiment, the gas control microfluidic device may also comprisea check valve to allow the gas to leave the microfluidic device.Further, the gas control microfluidic device may also comprise vacuumchannels. When vacuum is applied to the vacuum channels the microfluidicdevice may stretch to emulate cellular physiology in vivo. The gascontrol may also comprise sensors, such as oxygen sensors, in order tomonitor the gas levels within the microfluidic device.

In one embodiment, a microfluidic device is contemplated, comprising: a)one or more fluidic channels; b) gas channels around at least a portionof the perimeter of said one or more fluidic channels, separated fromsaid one or more fluidic channels by a gas-permeable wall. In oneembodiment, said microfluidic device comprises polydimethylsiloxane(PDMS). In one embodiment, said microfluidic device further comprises avalve in contact with said gas channels. In one embodiment, saidmicrofluidic device further comprises sensors. In one embodiment, saidgas channels are around the entire perimeter of said working channels.

The present invention contemplates, in one embodiment, a method ofcontrolling gas transport, comprising: a) providing a microfluidicdevice comprising i) one or more fluidic channels, and (ii) gas channelsaround at least a portion of the perimeter of said one or more fluidicchannels, separated from said fluidic channels by a gas-permeable wall;c) introducing a fluid into said one or more fluidic channels at a flowrate; b) introducing a non-oxygen gas into said gas channels as tocontrol the gas transport into said fluid. In one embodiment, saidmicrofluidic device comprises polydimethylsiloxane (PDMS). In oneembodiment, said microfluidic device further comprises a valve incontact with said gas channels. In one embodiment, said microfluidicdevice further comprises sensors. In one embodiment, said gas channelsare around the entire perimeter of said working channels.

Another embodiment contemplated to control gas within microfluidicdevices is using anaerobic cell culture incubators. A gas-permeablemicrofluidic device, may be controlled using a gas-controlled oranerobic cell culture incubator, in some embodiments in conjunction withperfusion manifold assemblies and culture modules. A silicone material,such as PDMS, allows rapid gas exchange between the channels within themicrofluidic device and the external environment of the microfluidicdevice, since the incubator volume/gas supply may be considered infinitecompared to the small volume of a microfluidic device. The incubatorconditions, in most instances, will define the gas microenvironmentexperienced by the cells within the microfluidic device, regardless offluid flow rate. There are generally three major sources for gastransport within a microfluidic device: incubator/environment air,dissolved gas in flowing fluid/media entering the microfluidic device,and cellular metabolism/processes.

Cellular gas uptake and release is an important factor of the gasmicroenvironment and differs between cell types. Oxygen delivery throughcell culture media alone is insufficient to maintain many cell types,thus the main oxygen source is the transport through a gas-permeablematerial. The maximum hepatocyte uptake rate in microfluidic devices,based on literature values and scaled to the cell culture area ofmicrofluidic device of U.S. Pat. No. 8,647,861, may be considered to be88 nmol/hr. Colonic oxygen uptake rate in microfluidic devices may beconsidered to be 2,020 nmol/hr, based on literature values and scaled tothe culture area of the microfluidic device of U.S. Pat. No. 8,647,861.However, oxygen delivery through media flow alone is only 5.8 nmol/hr,calculated based on the carrying capacity of water for oxygen and a flowrate of 30 μL/hr. Oxygen delivery through a PDMS microfluidic device,such as that present in U.S. Pat. No. 8,647,861, may be considered to be574 nmol/hr, which is a significant improvement on the oxygen deliveryrate of media flow alone. A system is contemplated, comprising agas-permeable microfluidic device having at least one channel, whereinsaid gas-permeable microfluidic device is disposed in a gaseousenvironment. In one embodiment, said gaseous environment is controlledby an incubator. In one embodiment, said gaseous environment iscontrolled by a hypoxic incubator. In one embodiment, said gaseousenvironment is a hypoxic environment. In one embodiment, said gaseousenvironment is a hyperoxic environment. In one embodiment, said systemis configured such that said gaseous environment controls the gasconcentration in said gas-permeable microfluidic device. In oneembodiment, said channel comprises a membrane. In one embodiment, saidchannel comprises cells.

In another embodiment, a method is contemplated of controlling the gasconcentration within a microfluidic device comprising: (i) providing amicrofluidic device comprising at least one channel; (ii) placing saidmicrofluidic device in a gaseous environment, such that said at leastone channel assumes the gas concentration of said gaseous environment.In one embodiment, said method further provides an incubator, andwherein said gaseous environment is controlled by said incubator. In oneembodiment, said incubator is a gas-controlled incubator. In oneembodiment, said gaseous environment is a hypoxic environment. In oneembodiment, said gaseous environment is a hyperoxic environment. In oneembodiment, said microfluidic device is gas-permeable. In oneembodiment, said channel comprises a membrane. In one embodiment, saidchannel comprises cells. In one embodiment, said cells compriseepithelial cells. In one embodiment, said cells comprise endothelialcells.

Definitions

The term “microfluidic” as used herein, relates to components wheremoving fluid is constrained in or directed through one or more channelswherein one or more dimensions are 1 mm or smaller (microscale).Microfluidic devices are described in the U.S. Pat. No. 8,647,861, andthe International Patent App. No. PCT/US2014/071611, the contents ofeach are incorporated herein by reference (such microfluidic devices arealso referred to herein as “chips”). Microfluidic channels may be largerthan microscale in one or more directions, though the channel(s) will beon the microscale in at least one direction. In some instances, thegeometry of a microfluidic channel may be configured to control thefluid flow rate through the channel (e.g. increase channel height toreduce shear). Microfluidic channels can be formed of various geometriesto facilitate a wide range of flow rates through the channels.

The phrases “connected to,” “coupled to,” “in contact with,” and “incommunication with” as used herein, refer to any form of interactionbetween two or more entities, including mechanical, electrical,magnetic, electromagnetic, fluidic, and thermal interaction. Forexample, in one embodiment, channels in a microfluidic device are influidic communication with a fluid source such as a fluid reservoir. Twocomponents may be coupled to each other even though they are not indirect contact with each other. For example, two components may becoupled to each other through an intermediate component (e.g. tubing orother conduit). Thus, a working fluid in a rigid container can be influidic communication with a working fluid reservoir via tubing or otherconduit.

The term “channels” as used herein, are pathways (whether straight,curved, single, multiple, in a network, etc.) through a medium (e.g.,silicon) that allow for movement of liquids and gasses. Channels thuscan connect other components, i.e., keep components “in communication”and more particularly, “in fluidic communication” and still moreparticularly, “in liquid communication.” Such components include, butare not limited to, liquid-intake ports and gas vents. Microchannels arechannels with dimensions less than 1 millimeter and greater than 1micron.

“Microchannels” are channels with dimensions less than 1 millimeter andgreater than 1 micron. Additionally, the term “microfluidic” as usedherein relates to components where moving fluid is constrained in ordirected through one or more channels wherein one or more dimensions are1 mm or smaller (microscale). Microfluidic channels may be larger thanmicroscale in one or more directions, though the channel(s) will be onthe microscale in at least one direction. In some instances, thegeometry of a microfluidic channel may be configured to control thefluid flow rate through the channel (e.g. increase channel height toreduce shear). Microfluidic channels can be formed of various geometriesto facilitate a wide range of flow rates through the channels.

The present invention contemplates a variety of “microfluidic devices,”including but not limited to microfluidic device, perfusion manifoldassemblies (without microfluidic devices), and perfusion manifoldassemblies engaged with microfluidic devices. However, the methodsdescribed herein for engaging microfluidic devices (e.g. by drop-to-dropconnections), and for perfusing microfluidic devices are not limited tothe particular embodiments of microfluidic devices described herein, andmay be applied generally to microfluidic devices, e.g. devices havingone or more microchannels and ports.

A surface or a region on a surface is “hydrophobic” when it displays(e.g. advancing) contact angles for water greater than approximatelyninety (90) degrees (in many cases, it is preferable that both advancingand receding contact angles are greater than approximately 90 degrees).In one embodiment, the hydrophobic surfaces of the present inventiondisplay advancing contact angles for water between approximately ninety(90) and approximately one hundred and ten (110) degrees. In anotherembodiment, hydrophobic surfaces have regions displaying advancingcontact angles for water greater than approximately one hundred and ten(110) degrees. In another embodiment, hydrophobic surfaces have regionsdisplaying receding contact angles for water greater than approximately100 degrees. It is important to note that some liquids, and particularlysome biological liquids, contain elements that may coat a surface afterwetting it, thereby affecting its hydrophobicity. In the context of thepresent invention, it may be important that a surface resists suchcoating from a liquid of intended use, for example, that such coatingdoes not create an advancing and/or receding contact angle that is lessthan 90 degrees over the duration that the surface remains wetted by thesaid liquid.

A surface or a region on a surface is “hydrophilic” when it displays(e.g. advancing) contact angles for water less than approximately ninety(90) degrees, and more commonly less than approximately seventy (70)degrees (in many cases it is preferable that both the advancing andreceding contact angles are less than approximately 90 degrees orapproximately 70 degrees).

Measured “contact angles” can fall in a range, i.e. from the so-calledadvancing (maximal) contact angle to the receding (minimal) contactangle. The equilibrium contact is within those values, and can becalculated from them.

Hydrophobic surfaces “resist wetting” by aqueous liquids. A material issaid to resist wetting by a first liquid where the contact angle formedby the first liquid on the material is greater than 90 degrees. Surfacescan resist wetting by aqueous liquids and non-aqueous liquids, such asoils and fluorinated liquids. Some surfaces can resist wetting by bothaqueous liquids and non-aqueous liquids. Hydrophobic behavior isgenerally observed by surfaces with critical surface tensions less than35 dynes/cm. At first, the decrease in critical surface tension isassociated with oleophilic behavior, i.e., the wetting of the surfacesby hydrocarbon oils. As the critical surface tensions decrease below 20dynes/cm, the surfaces resist wetting by hydrocarbon oils and areconsidered oleophobic as well as hydrophobic.

Hydrophilic surfaces “promote wetting” by aqueous liquids. A material issaid to promote wetting by a first liquid where the contact angle formedby the first liquid on the material is less than 90 degrees, and morecommonly less than 70 degrees.

As used herein, the term “shear stress” in general refers to an appliedforce per unit area, acting parallel to a surface element. “Shear” or“shear stress” refers to a force on an object parallel to the face of anobject. Shear stress is primarily caused by friction between fluidparticles, related to fluid viscosity, and a component of shear strain.τ (Greek: tau) refers to a combined effect of viscosity and relativevelocities where the stress is parallel to the surface of the material,as opposed to normal stress when the stress is perpendicular to thesurface. Shear stress is relevant to the motion of fluids upon surfaces,which result in the generation of shear stress. the shear stress (σ). Asan example, fluid flow across the surface of a cell may exert shearstress on said cell.

As used herein, the term “shear rate” or “shear strain” refers to therate of change of velocity at which one layer of fluid passes over anadjacent layer. “Shear rate” is also referred to as γ, (Greek: gamma G)or “rate of shear”. In a non-Newtonian fluid, such as blood, therelationship between shear stress and shear rate is different.

A “gas exchanger” refers to a mechanical or chemical component, such ascomprised within a microfluidic device, which allows the transport ofgas. The gas exchanger may alternatively be known as a “gas transportmembrane,” “gas exchange membrane,” or “gas control membrane.”

“Metabolism” is a chemical process that occurs within a living organism,such as a cell, in order to maintain life. “Cellular metabolism” is“metabolism” specific to cells. For example, a cell may metabolize apharmaceutical compound.

A “manifold” is a physical component of a system that takes in a fluid(gas or liquid) and splits the flow of that fluid into multiple flowroutes. An example of a “manifold” is a pipe, chamber, or channel thatbranches into several new openings.

The phrase “flow of a medium” means bulk movement of a fluidic mediumprimarily due to any mechanism other than diffusion. For example, flowof a medium can involve movement of the fluidic medium from one point toanother point due to a pressure differential between the points. Suchflow can include a continuous, pulsed, periodic, random, intermittent,or reciprocating flow of the liquid, or any combination thereof. Whenone fluidic medium flows into another fluidic medium, turbulence andmixing of the media can result. It may be advantageous to usereciprocating flow, meaning that a volume of fluid is alternatelyintroduced into and then withdrawn from the microfluidic device or froma portion of the microfluidic device, such as a channel or chamber. Insuch cases, the reciprocating flow may be driven by a device that isconnected or in fluidic communication with the microfluidic device orwith a portion of the microfluidic device.

As used herein, the terms “molecules,” “particles,” and “particulates”refers broadly to a constituent of matter, both viable and non-viable.As one example, a particle refers to a cell, such as a cell within afluid, including both cells normally present in the blood of healthypatient (white cell, red cell, platelets, etc.), cells not normallypresent into the bloodstream such as circulating tumor cells. However,the fluid is not limited to blood, i.e. cells are found in fluids, suchas macrophages found in lung fluid, etc. As another example, a particlerefers to microorganisms, e.g., spores, virions, bacterium, such asfound in normal flora or present in diseased states, and microscopicphysical particles/particulates, including but not limited topollutants, as well as any physical particles/particulates that couldenter the blood stream or other bodily fluid. Particles also includebeads and the like, which can be conveniently used in some embodimentsin place of cells in order to take measurements or otherwise evaluate aparameter, e.g. flow rate, buoyancy, viscosity, shear, etc.

The term “small molecule” refers to a molecule below the molecularweight of 1 kDa.

A “xenobiotic” is a molecule that does not typically occur in the humanbody, and may be considered a chemical. Xenobiotics are typicallysmaller than biologics, which are typically found to be naturallyoccurring the human body. A xenobiotic is generally below the size of 1kDa.

The term “rigid,” when applied to polymers, refers to a polymer with amodulus of elasticity, or Young's Modulus, or flexural modulus, above0.1 GPa.

The term “elastomeric” or “flexible,” when applied to polymers, refersto a polymer with a modulus of elasticity, or Young's Modulus, orflexural modulus, below 0.1 GPa.

The term “gas-permeable” refers to a polymer which largely allows thetransport of gases through its material makeup.

The term “gas-impermeable” refers to a polymer which does not largelyallow the transport of gases through its material makeup.

The term “low-absorbing” refers to a polymer which does not largelyallow for the absorption of xenobiotics or small molecules into itsmaterial makeup.

The term “absorbing” refers to a polymer which does largely allow forthe absorption of xenobiotics or small molecules into its materialmakeup. The term “solid substrate” as used herein, refers to a substratethat may be biological, nonbiological, organic, inorganic, or acombination of any of these, existing as particles, strands,precipitates, gels, sheets, tubing, spheres, containers, capillaries,pads, slices, films, plates, slides, etc. The solid substrate ispreferably flat but may take on alternative surface configurations. Forexample, the solid substrate may contain raised or depressed regions,such as microfluidic channels and/or inlet and outlet ports. Forexample, the substrate may be functionalized glass, Si, Ge, GaAs, GaP,Sioxygen, SiN4, modified silicon, nitrocellulose and nylon membranes, orany one of a variety of gels or polymers such as(poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene,polycarbonate, or combinations thereof. Other suitable solid substratematerials are be readily apparent to those of skill in the art. Thesurface of the solid substrate may also contain reactive groups, whichcould be carboxyl, amino, hydroxyl, thiol, or the like. More preferably,the surface will be optically transparent and will have surface Si—OHfunctionalities, such as are those found on silica surfaces.

The term “porous membrane” as used herein, refers to a material that isflexible, elastic, or a combination thereof with pores large enough toonly permit exchange of gases and small chemicals, or large enough topermit migration and transchannel passage of large proteins, and/orportions thereof. The membrane may also be designed or surface patternedto include micro and/or nanoscopic patterns therein such as grooves andridges, whereby any parameter or characteristic of the patterns may bedesigned to desired sizes, shapes, thicknesses, filling materials, andthe like.

The term “chamber” as used herein, refers to an isolated region of amicrochannel that is separated by a porous membrane. For example, theporous membrane may extend longitudinally down the midpoint of amicrochannel thereby providing an upper chamber and a lower chamber.

The term “media” refers to a liquid for conveying a substance. In oneembodiment, the substance is nutritive, such as in a culture medium.

The term “valve” refers to a mechanical component that can control fluidflow. Diaphragm valves (or membrane valves) consists of a valve bodywith two or more ports, a diaphragm, and a “weir or saddle” or seat uponwhich the diaphragm closes the valve.

As used herein, the term “rheology” refers to the flow and deformationof fluids, gases and solids under the influence of mechanical forces. Inother words, rheology may be referred to as physics relating tonon-Newtonian flow and Newtonian flow of liquids, soft solids, solidsand gases.

As used herein, the term “biomimetic” or “biomimicry” refers tomaterials, e.g. fluids, membranes, etc., synthetic systems, syntheticdevices, machines etc., that have functions that mimic a biologicalprocess or biological component, e.g. blood, intestinal contents, lungfluid, etc.

The term “transparent,” generally refers to the ability of light to passthrough. For example, a microfluidic device may be consideredtransparent if light is able to pass through the body of themicrofluidic device and the contents of the channels may be able to beseen by a standard light microscope.

The term “crash out” or “crashing out of solution” refers to when theconcentration of a solute or compound in a solution reaches a pointwhere the solute or compound precipitates out of the solution, forming asolid.

It is not intended that the present invention be limited by the natureof the indentations. The term “indentation” as used herein, refers to aspace, cavity, dent, crater, well, depression, hollow, recess orimpression that is formed in the surface. In a preferred embodiment,indentations do not extend through the entire thickness of a surface.While a hole can be an indentation, the hole preferably does not extendcompletely through the surface. In one embodiment, each of saidindentations has a depth that extends up to the midpoint of said firstor second element (i.e. the depth of the indentation is equal to or lessthan one-half the thickness of the surface). In one embodiment, saidsecond surface is crenellated and the gaps comprise said indentations.In another embodiment, the indentations have raised edges. The term“raised edge” means that the edge of the indentation rises above theplane of the surface. In one embodiment, there are particles in theindentations (e.g. beads). It is not intended that the present inventionbe limited by the manner in which the indentations are manufactured. Inone embodiment, the indentations are introduced into the surface bytreating the surface (e.g. etching a surface of glass, silicon orotherwise etchable surface). In another embodiment, the indentations areintroduced by casting or molding. In a preferred embodiment, theindentations are integrally molded using a polymeric surface (e.g.plastic). The term “integrally molding” as used herein refers to themethod of casting such that features are of unitary construction. Theterm “unitary construction” refers to an association of elements (e.g.the surface and the indentations) such that they are formed from thesame piece of raw material without the need for further integration. Inone embodiment, the first surface comprises plastic and hasindentations. In one embodiment, said first surface is elastomeric.

As used herein, the term “gas transport” refers to the passage of a gasthrough a material or the passage of gas from one area to another. The“rate of gas transport” refers the measure of flow of gas through amaterial or from one area to another. The rate may refer to eithervolumetric flow rate or mass flow rate. Volumetric flow rate is thevolume of fluid which passes per unit time. Mass flow rate is the massof a substance which passes per unit of time.

The term “diffusion” refers to the movement of molecules or atoms from aregion of higher concentration to a region of lower concentration.

The term “gradient” refers to an increase or decrease in the magnitudeof a property. For example, a chemical potential gradient would be thechange in chemical potential across a system.

The term “adsorption” refers to the process by which a solid holds themolecules of a gas or liquid or solute as a film on its surface.

As used herein, the term “fluid” refers to either a liquid or gas, whichis unable to hold a fixed shape and yields easily to external pressure.As used herein, the term “fluid flow” refers to the movement of a fluid.As used herein, the term “fluid flow rate” refers to the measure of flowof a fluid.

A channel may be considered “open” if it lacks at least one wall in atleast one portion of the channel. Likewise, an open channel may be“capped” or covered with another object or material.

As used herein, the term “porosity” refers to the quality of beingporous, or comprising holes. The term “pores” refers to those holes. Theterm “porous elements” refers to components of a system which comprisepores, and therefore have a porosity.

A “cap” is an object or material that covers or “caps” another. A“capping layer” is a sheet or film which covers an invention component.

A “wall,” such as a “channel wall,” is a barrier or enclosure of ahollow area.

As used herein, the term “surface” refers to the outermost portion of anobject, such as the portion of an object in contact with anothercomponent. For example, a fluid may contact the surface of a channel,wherein that surface is a wall of the channel.

As used herein, the term “layer” refers to a flat or thin component,material, or object. For example, a sheet or film may be referred to asa layer.

As used herein, the term “side” refers to a surface which generallyopposes another surface. A “surface” may also be a position to the leftor right of an object or central point.

As used herein, the term “membrane” refers to a component or materialwhich allows the passage of molecules, fluids, cells, etc. through it

As used herein, the term “sheet” refers to a thin material. As usedherein, the term “film” also may refer to a thin material. The termssheet and film are interchangeable herein. The term “film” may alsorefer to a very thin liquid or layer of biological material, such asbacteria. The term “thin film” refers to a film of remarkable thinness,such as below 10 μm.

The term “maintain” refers to keeping a commodity or variable, such as arate of gas transport or a flow rate of media, relatively constant.

The term “resistant” refers to a material or component that is generallyimpervious to manipulation by another substance. However, the termresistant is qualified as largely impervious, such that the material orcomponent is not manipulated by the majority of another substance. Thereexists no entirely perfect material or structure. For example, a polymermay be resistant to small molecule absorption. The resistance of thatpolymer is compared to polymers that are known to absorb.

The term “cells” refers to the smallest structural and functional unitof an organism, such as a human. Cells may be “cultured” or grown on asurface or in an environment, such as within a microfluidic device.

A “gas concentration profile” refers to the gradient of gas molecules involume or the curve that results when the concentration of a gas isplotted versus position in that volume.

Liver oxygen zonation is the gradient of oxygenation within a liverenvironment. That gradient may be divided or designated to levels, suchthat some are considered aerobic and some are considered anaerobic.

As used herein, the term “hypoxic” refers to an environment that is lowin oxygen.

As used herein, the term “lumen” refers to the inside space of a tubularstructure.

As used herein, the term “contact” refers to one material, substance,fluid, object, etc. touching another. For example, a fluid in a channelcontacts the walls of the channel. For example, a tube may contact theinlet of a microfluidic device.

As used herein, the term “substantially free” refers to an environmentthat has a low concentration of a substance, such as a molecule, fluid,a particular gas, etc.

As used herein, the term “penetrate” refers to one material filling orentering another.

As used herein the term “cure” or “cured” refers to the solidification,toughening, hardening, and/or cross-linking of a material, such as apolymer. Curing may be initiated by heat, radiation, electron beams,chemical additives, the absence of a particular compound or gas, etc.Conversely, the term “uncured” refers to a material that has not yetbeen cured, solidified, toughened, hardened, and/or cross-linked.

The term “coating” refers to one material or substance that coversanother. The term “biological coating” refers to a coating that isbiological in nature or serves a biological purpose, such as interactswith cells, etc.

The term “excess” refers to a surplus of a material, component orsubstance.

The term “fabricate” refers to the creation, building, construction ormanufacturing of a component, such as to fabricate a microfluidicdevice.

As used herein, the term “by volume” or “percent by volume” refers to ameasure of a value, such as concentration, with regards to the totalvolume of a body or solution.

As used herein, the term “experimental protocol” refers to thedirections on how to set up and conduct an experiment.

As used herein, the term “modified experimental protocol” refers to anexperimental protocol that has been altered from its original form.

As used herein, the term “setup” refers to the way in which equipment orexperiments are organized, planned and/or arranged.

As used herein, the term “intended study” refers to an experimentalprotocol, also known as a study, as it is proposed to be run orexecuted.

As used herein, the term “infusion tubing” refers to tubing used toadminister intravenous drugs.

As used herein, the term “syringe” refers to a piece of medical andexperimental equipment comprising a nozzle and piston or bulb used suckin and eject liquid in a stream.

As used herein, the term “biological elements” refers to components ofan invention which are biological in nature such as cells orextracellular matrix.

As used herein, the term “taking actions” refers to active steps takenby the user of a method.

As used herein, the term “concentration” refers to the amount of asubstance, such as a compound or drug, per defined space. The term“apparent concentration” refers to the concentration of a substancewithout taking into account variability, such as absorption into thesystem.

As used here, the term “input” refers to the inlet of a system or thatwhich is put into a system.

As used herein, the term “output” refers to the outlet of a system orthat which exits a system.

As used herein, the term “sample” refers to a small quantity intended toextract information about that which the sample was removed from. As anexample, a sample may be taken from a fluidic experiment to be assayed.

As used herein, the term “effluent” refers to fluid which has flowed outof a system. The term “effluent sample” refers to a sample taken fromeffluent. The terms “effluent” and “effluent sample” may beinterchangeable.

The term “influent” refers to fluid which is to be flowed into a system.The term “influent sample” refers to a sample taken from influent. Theterms “influent” and “influent sample” may be interchangeable.

The term “quantification” refers to the counting and measuring ofobservations into quantities. For example, the concentration of acompound in a fluid may be quantified to make a quantification.

The term “percent absorption” refers to the percent of a compound orsubstance that absorbs into the system which it contacts.

As used herein, the term “assay” refers to the qualitative orquantitative measuring of the presence, amount, or function activity ofa target entity, such as an analyte. The term “assaying” refers to theaction of taking an assay of a target entity.

The term “analyte” refers to a substance whose chemical constituents maybe identified and/or measured.

As used herein, the term “metabolite” refers to a substance formed in ornecessary for metabolism. The term “apparent metabolite” refers to ametabolite as it seems to be, without taking into account variability,such as absorption into a system.

As used herein, the term “metabolism” refers to the chemical processesthat occur within a living organism in order to maintain life.

The term “scaffold” refers to a material that have been engineered topositively interact with biology, whether that biology is in vivo or invitro. For example, a membrane may be considered a scaffold.

As used herein, the term “variability” refers to the total variationseen in an experiment and can come from a variety of sources, including,but not limited to, process and biological population inconsistency.

As used herein, the term “introduce” refers to the input of a fluid ormaterial or biological element, etc., into a system.

As used herein, the term “determine” refers to the active step ofascertaining or establishing a next step, result, etc.

As used herein, the term “error bars” refer to the line through a pointon a graph, parallel to one of the axes, which represents theuncertainty or error of that data. The inventions presented herein maybe able to either reduce error bars or aid scientists in making them foraccurate.

As used herein, the term “half maximal inhibitory concentration (IC₅₀)”refers to a measure of the potency of a compound in inhibiting aspecific biological or biochemical function. In other words, the IC₅₀ isa quantitative measure of how much of a compound is needed to inhibit abiological process. The IC₅₀ represents the concentration of a compoundthat is needed for 50% inhibition or maximum effect in vitro, such as ina microfluidic device. For example, if the intended study shows an IC₅₀at a dosing concentration of 1 μM, the results of the compounddistribution kit may indicate that the actual IC₅₀ is at an exposureconcentration in the range of 0.6 μM to 1 μM.

As used herein, the term “threshold” refers to a magnitude that theresult of a certain reaction, phenomenon, result or condition may becompared to. For example, if compound absorption is over a certainthreshold, the intended study may not be worth completing.

As used herein, the term “time point” refers to a point during anexperiment or clinical procedure when an action is taken.

As used herein, the term “measurement” refers to the assignment of anumerical characteristic to an object or event. For example, ameasurement of system absorption or compound distribution in a systemmay be taken.

As used herein, the term “ratio” refers to the quantitative relationbetween two amounts showing the number of times one value contains or iscontained in the other. Ratios are helpful in comparison.

As used herein, the term “fluidic circuit” refers to a system ofconnections, including tubing, channels, conduits, arteries, veins,chambers, tanks, ducts, grooves, mains, passages, troughs, pipes,conduits, inlets, outlets, etc. that a fluid may flow through. Is notintended that a fluidic circuit is limited to a continuous system. Afluidic circuit may comprise breaks in flow, such as valves, bubbles, orempty spaces.

As used herein, the term “chromatography” refers to the separation of amixture by passing it through in solution or as a vapor through a mediumin which the components move at different rates.

The term “spectrometry” refers to the separation and measurement ofspectral components of a physical phenomenon. As used herein,spectrometry may be used to quantify analytes and/or metabolites in asystem.

As used herein, the term “drug” refers to a medicine or other substancewhich has physiological effects when introduced to a biological system.The term “pharmaceutical” refers to drugs for medical purposes. The term“drug candidate” refers to a molecule that has been shown to havesufficient target selectivity and potency, and favorable medicine-lineproperties and justifies further development.

As used herein, the term “compound” may refer to any chemical,biological and/or pharmaceutical substance composed of many molecules ingaseous, liquid, or solid form.

Herein, the terms “substance” refers to a matter with uniformproperties.

As used herein, the term “compound distribution” refers to theconcentration of a compound across a system.

As used herein, the term “polymer” refers to a substance that has amolecular structure consisting chiefly or entirety of a large number ofsimilar units bonded together.

As used herein, the term “plastic” refers to a synthetic material madeup polymers. A plastic is a type of polymer.

As used herein, the term “regulate” refers to the control orcoordination of a process and/or system.

As used herein, the term “fill” refers to when an empty space isoccupied by matter.

As used herein, the term “runs along the length” refers to when onematerial, substance, component, etc. is placed in coordination with thelength of another material, substance, component, etc. For example, achannel may run along the length of a substrate.

As used herein, the term “constant” refers to an action and/or processthat occurs continuously over a period of time.

As used herein, the term “sensor” refers to a component which detects ormeasures a physical property. Example of sensors include flow ratesensors, gas sensors, fluorescent sensors, etc.

The term “oxygen sensor” refers to a sensor which detects the presenceor measures the concentration of oxygen.

As used herein, the term “mechanical stability” refers to the physicalstrength of a material and/or component.

As used herein, the term “at least partially” refers to an extent whichranges from only in part to full.

As used herein, the term “border” refers to the edge or boundary or partnear. A component may border another component. A component may alsohave a border.

As used herein, the term “enclose” refers to a component or material orsubstance which surrounds another. A component may be partiallyenclosed, or not entirely surrounded.

The term “reservoir” or “fluidic reservoir” refers to a container wherefluid collects.

The term “fluidic communication” refers to continuous fluid contact in asystem. For example, two components may be in fluidic communication ifthey each comprise a fluid, and those fluids are in contact.

The term “backplane” refers to a material or component used formechanical stability. A backplane may have additional features supportedon it, such as protrusions or channels.

The term “gasket” or “gasketing layer” refers to a material or componentused for sealing a junction between two surfaces. For example, agasketing layer may be used between a fluidic backplane and a reservoir.

The term “projecting member” refers to a component that protrudes fromthe body of a system or device.

The term “cap” or “capping layer” refers to a material or component usedfor topping or covering. For example, channels in a backplane may becapped or covered with a capping layer.

The term “media” refers to a liquid for use in a biological system. Forexample, when cell media is oftentimes needed when culturing cells. Theterm “culture media” refers to media being used to culture biologics,such as cells or bacteria. The term “cell media” or “cell culture media”refers to media used for culturing cells in particular.

As used herein, the term “additives” refers to a substance added tosomething in order to improve it, preserve it, or otherwise. Forexample, additives may be added to culture media in order to have bettergrowth.

The term “solvent” refers to a fluid that may be used to dissolve othersubstances or fluids or solutes.

The term “solute” refers to a substance, such as an additive, that maybe dissolved in a solute.

The term “modulus of elasticity”, also known as flexural modulus, alsoknown as Young's Moduli, refers to the measurement of an object ormaterial or substance's resistance to being deformed elastically(non-permanently) when a stress is applied to it. Polymers may be gaugedas rigid or elastomeric based on their modulus of elasticity. Herein,any polymer with a modulus of elasticity over 0.1 GPa is consideredeffectively rigid, or non-flexible, certainly for the purposes ofmicrofluidic device fabrication. Rigid polymers may fall in the range of0.1 GPa to 150 GPa. Metals usually have a modulus of elasticity value ofat least 30 GPa or greater. For example, aluminum can have a modulus ofelasticity value up to about 69 GPa.

Herein, the term “recirculation” refers to the process of circulating afluid again through a system, such as a fluidic circuit.

The term “recirculation pathway” refers to a fluidic circuit or fluidicpathway or system of tubing and/or channels used for recirculation.

Herein, the term “reciprocation” refers to a process in a fluidic systemwhere the fluid is flowed back and forth through said system.

The term “reciprocation pathway” refers to a fluidic circuit or fluidicpathway or system of tubing and/or channels used for reciprocation.

The term “reciprocation actuator” refers to a mechanical component oractuator used to change the direction of a fluid during reciprocationand/or in reciprocation pathway.

Herein, the term “actuator” refers to mechanical component that isresponsible for moving and controlling a mechanism of a system.

Herein, the term “body” refers to the physical structure of a system ordevice. For example, the body of a microfluidic device is the mainstructure of the device, for example built out of a polymer.

Herein, the term “viability” refers to the ability to function or workcorrection. For example, the viability of culture cells may be qualifiedby the cells having the correct morphology, forming uniform monolayers,expressing the correct genes and markers, outputting correct levels ofmetabolites, etc.

The term “oxygen-carrying component” refers broadly to a substancecapable of carrying oxygen. In one embodiment, the oxygen-carryingcomponent is native or modified hemoglobin. As used herein, the term“hemoglobin” refers to the respiratory protein generally found inerythrocytes that is capable of carrying oxygen. Modified hemoglobinincludes, but is not limited to, hemoglobin altered by a chemicalreaction such as cross-linking, polymerization, or the addition ofchemical groups (e.g., polyethylene glycol, polyoxyethylene, or otheradducts). Similarly, modified hemoglobin includes hemoglobin that isencapsulated in a liposome. For example, the hemoglobin may be derivedfrom animals and humans; preferred sources of hemoglobin are cows andhumans. In addition, hemoglobin may be produced by other methods,including recombinant techniques. A most preferredoxygen-carrying-component of the present invention is “polyethyleneglycol-modified hemoglobin.” The term “polyethylene glycol-modifiedhemoglobin” refers to hemoglobin that has been modified such that it isassociated with polyethylene glycol; generally speaking, themodification entails covalent binding of polyethylene glycol (PEG) tothe hemoglobin.

The term “non-oxygen-carrying component” refers broadly to substanceslike plasma expanders that can be administered, e.g., for temporaryreplacement of red blood cell loss. In preferred embodiments of theinvention, the non-oxygen-carrying component is a colloid (i.e., asubstance containing molecules in a finely divided state dispersed in agaseous, liquid, or solid medium) which has oncotic pressure (colloidosmotic pressure prevents, e.g., the fluid of the plasma from leakingout of the capillaries into the interstitial fluid). Examples ofcolloids include hetastarch, pentastarch, dextran-70, dextran-90, andalbumin.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used to describe the present invention,in connection with percentages means ±5%.

As used herein, the term “substantially” is a relative term that can beused to indicate similar dimensions (e.g. height, width, etc.) orsimilar features (e.g. porosity, linearity, etc.) that need not beidentical to a reference, e.g. preferably at least 80% of the dimensionor feature, more typically, at least 90%, or at least 95%, or at least97% or at least 99% or more.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts one embodiment of a low-absorbing, gas-permeablemicrofluidic device comprising a gas exchanger.

FIG. 2 depicts an exploded view of one embodiment of a low-absorbing,gas-permeable microfluidic device. The exploded view shows that themicrofluidic device comprise a gasket, a top channel layer, a membrane,a bottom channel layer, a top channel, a bottom channel and agas-exchanger.

FIG. 3 shows a cross-sectional view of one embodiment of alow-absorbing, gas-permeable microfluidic device comprising a gasket, atop layer, a membrane, a bottom layer, a top channel, a bottom channel,and a gas-exchanger.

FIG. 4 shows an absorbing, gas-permeable microfluidic device fabricatedfrom PDMS.

FIG. 5 shows a low-absorbing, gas-impermeable microfluidic devicefabricated from COP and SEBS gasketing layers.

FIG. 6 shows an exploded view of a low-absorbing, gas-impermeablemicrofluidic device fabricated from COP and SEBS comprising gaskets, atop channel layer, a cell culture membrane, and a bottom channel layer.

FIG. 7 shows one embodiment a perfusion manifold assembly comprisingseparate gasketing and capping layers. The embodiment of the perfusionmanifold assembly also comprises a lid, different varieties of filters,a lid gasket, reservoirs, a fluidic backplane, a skirt and screws.

FIG. 8 shows a perfusion manifold assembly comprising one gasketing andcapping layer. The perfusion manifold assembly also comprises a lid,different varieties of filters, a lid gasket, reservoirs, a fluidicbackplane, a skirt and screws.

FIG. 9 depicts the drug development triangle, comprising importantaspects of developing an understanding of how a therapeutic is going tointeract with the body. In summary, the study of pharmacokinetics aimsto understand how and quantitatively predict how a particular dose ormass of compound is processed by the various organs in the body toproduce and exposure concentration. Pharmacodynamics aims to understandand predict how that exposure concentration results in a given effect(either efficacy or toxicity). Organ-Chips can, have, and are being usedto study both pharmacokinetics and pharmacodynamics, which underscoresthe importance of understanding and controlling the concentration ofcompounds in microfluidic devices since concentration it is vital forboth fields, and, therefore, vital for understanding and predicting howa pharmaceutical is going to interact with the human body.

FIG. 10A depicts the absorption of a small molecule (Bupropion) invarious embodiments of the microfluidic system comprising of amicrofluidic device and perfusion manifold assembly, while 10B depictsthe results of a test of that same compound in the same setup for livermetabolism by the metabolizing enzyme CYP2B6. The apparent metabolism ofdrug by liver cells in both an absorbent microfluidic device fabricatedfrom PDMS and a gas-impermeable, low-absorbing microfluidic devicefabricated from COP are depicted, demonstrating the effects ofabsorption on the apparent rate of metabolism, when quantified byproduction of a metabolite. It can be seen that the highly absorbingsystems results in greater under-prediction of metabolism than thenon-absorbing and lower-absorbing systems.

FIG. 11 depicts the compound distribution profile in a high-absorbing,gas-permeable microfluidic device fabricated out of PDMS. The modeldepicts a highly absorbing compound, midazolam, being perfused throughboth the top and bottom channels of the microfluidic device at 150uL/hr.

FIG. 12 depicts a drug response curve and the influence of absorption onit. Absorption causes the observed dose response curve to shift as theexposure concentration of the drug to the cells (X-axis) is modulateddue to absorption.

FIG. 13A-B illustrates the test protocol for a time-dependent materialabsorption test (including absorbing materials such as PDMS). This studyaims to determine the intrinsic material-compound interaction propertiesof drug-absorbing materials, PDMS or otherwise.

FIG. 14 depicts a fine element analysis, or a computational model, ofrecovered compound concentration from a set volume of PDMS afterdifferent time points for compounds of varying diffusivity. Data fromtime-dependent material absorption tests, like those depicted in FIG.13A-B, is compared to graphs like the one depicted here and used todetermine the fundamental parameters defining compound-specificabsorption into the material tested; here, a determination ofdiffusivity, or speed of compound loss.

FIGS. 15A and 15B depicts the absorption of the drug Diazepam into bothmaterials PDMS and COP over time, based on the recovered concentrationof Diazepam remaining in the fluid contained in the glass vials wherethe material is contained. This depicts compound “loss” to the materialover time.

FIG. 16 depicts protein binding or “loss” of compound to proteinssuspended in the cell culture media. As more protein is added to themedia, in this case in the form of increasing concentrations of fetalbovine serum (FBS), there is additional loss of compound.

FIG. 17 shows a computational absorption model of a microfluidic devicecomprising a top channel, a bottom channel, and a membrane separating atleast a portion of said top channel (3) and bottom channel was built.The model allows different variables to be changed, includingpermeability of the material, absorbance of the material, flow rate ofthe fluid in the top and bottom channels, diffusivity of the compound inthe fluid, etc.

FIGS. 18A and 18B depict a comparison between computational modelprediction of microfluidic device absorption and the results ofexperiments for the test compound Coumarin. Coumarin was flowed througha high-absorbing, gas-permeable microfluidic device fabricated out ofPDMS and the recovered concentration in the bottom and top channels weresampled. The experiment was run at two different flow rates, 60 μL/hr asseen in FIG. 18A and 150 μL/hr as seen in FIG. 18B. These results wereplotted vs the output of COMSOL models of the microfluidic device, asdescribed above, with the measured material absorption parameters forCoumarin and the two flowrates flowrate as model inputs. The data andmodels are in good agreement.

FIG. 19 depicts the complexity of modeling and understanding thedynamics of compound disposition in the interior of an absorbingmicrofluidic device, even in the absence of absorption. This includesbiological/physiological factors such as passive cellular permeability,metabolism and transport across the membrane.

FIGS. 20A and 20B depict the results of absorption testing of manydifferent small molecule compounds and the relationship between thephysicochemical parameter “log P” or octanol partitioning and “log K” orPDMS partitioning. Note that an R² value of 0.515 indicates a weakcorrelation between the two parameters. Taken alone, log P cannot beused to predict PDMS partitioning. When absorption is considered withrespect to both log P and molecular weight simultaneously, we see evenless of a correlation between a binary “will/will not” absorb and thesetwo parameters.

FIGS. 21A and 21B depict the results of absorption testing of individualdrug molecules, Drug 36 and Drug 48, respectively.

FIG. 22 shows a depiction of a 2D microfluidic device computationalmodel for the use of running microfluidic device studies in silico,complete with the barrier created by the cell layer and cellularprocesses, like metabolism, included. These models can be used to designcell-based microfluidic device experiments based on material propertiesand expected rates of the cellular processes, including to designexperiments to minimize the effects of absorption. When run in thismanner, the models output the expected experimental result (e.g.microfluidic device effluent concentrations of a dosed compound).Conversely, the models can be run in some cases after experimental datais generated to “correct” for the contribution of compound loss due toabsorption. For example, liver cell metabolism results in compound lossmuch like PDMS absorption causes compound loss. Given the intrinsicmaterial properties, the amount of compound expected to be lost to PDMSabsorption can be “subtracted out” from the total compound lost to bothabsorption and cellular metabolism in order to deduce the rate ofcellular metabolism.

FIG. 23, 23.1-23.4 shows a table of polymers and a listing of theircharacteristics, most notably their flexural modulus or modulus ofelasticity. A number of polymers in the table have been highlighted asexhibiting elastomeric properties. Those polymers that may be consideredelastomeric have a modulus of elasticity under 0.1 GPa. Chlorinated(polyvinyl chloride) PVC has also been marked as elastomeric due to itssurface hardness.

FIG. 24 shows a chart of different embodiments of the present inventionand the problems that lead to their invention. The elastomericmicrofluidic device, which is high-absorbing and gas-permeable, has beendescribed in U.S. Pat. No. 8,647,861. It was noted that thismicrofluidic device fabricated from an elastomeric polymer (PDMS) wasboth highly gas-permeable and prone to absorption of small molecules orxenobiotics. In some instances, the microfluidic device was toogas-permeable. In other words, the body and channels of the microfluidicdevice were too susceptible to the gas concentration of the ambientenvironment due to the permeability of the elastomer. Resulting fromthat discovery two different embodiments were invented. The first was anelastomeric microfluidic device with gas channels running around workingchannels as shown in FIGS. 93 and 94. A gas, such as nitrogen, could beflowed through the gas channels in order to transport that gas into theworking channels. Because the walls separating the gas channels from theworking channels are highly gas-permeable, the gas channels act to setthe oxygen concentration of both the bulk of the microfluidic device;the channels could be depleted of oxygen, by flowing nitrogen throughthe gas channels, for example, or any other gas for that matter.Alternatively, the gas channels may also, in one embodiment, worktowards introducing more oxygen into the microfluidic device, such thatthe concentration of oxygen in the microfluidic device is higher thanthe ambient environment. The second embodiment resulting from the highpermeability of the elastomeric, high-absorbing, gas-permeablemicrofluidic device was to contact the outside surfaces of themicrofluidic device with a thin film or mask of rigid or gas-impermeablepolymer in order to limit gas transport through the bulk of themicrofluidic device. Resulting from the discovery that the microfluidicdevice of U.S. Pat. No. 8,647,861 was highly prone to absorption ofsmall molecules an embodiment of a low-absorbing, gas-impermeablemicrofluidic device was fabricated from rigid materials. It was thendiscovered that the low-absorbing, gas-impermeable microfluidic devicewas too gas-impermeable for some experiments. One embodiment to overcomethe gas-impermeability was to add supplements to the media or fluid,such as to augment (e.g. increase) the gas carrying capacity of themedia or fluid. It was found, however, that these supplements aresometimes difficult to work with. Another embodiment to overcome thegas-impermeability was to flow fluids or media at high flow rates inorder to introduce a higher concentration of dissolved oxygen into thechannels of the microfluidic device. Unfortunately, there are somedisadvantages to high flow rates including fluid or media waste. In thecases that cells are cultured in the microfluidic device, importantcellular signals can be washed away. Further, higher flow rates resultin higher levels of shear which may not always be favorable. In order toovercome these disadvantages, fluid or media may be recirculated.Sometimes, though, recirculation setups can be bulky and requireequipment that is difficult to use. In those cases, fluid may bereciprocated, or flowed back and forth through the device. Reciprocationis non-obvious in the case of studying cells in vitro as fluid in vivodoes not flow two ways. A surprising discovery was that cells in vitrodisplayed high levels of viability and organ-specific function withreciprocated media. Finally, another solution to the gas-impermeabilityof rigid microfluidic devices was to introduce a gas exchanger to themicrofluidic device. In one embodiment, the gas exchanger could be builtfrom a material such as Teflon (PTFE). However, materials such as Teflonare oftentimes difficult to bond or are not transparent. In oneembodiment, the gas exchanger comprises a thin piece ofpolydimethylsiloxane (PDMS). However, thin pieces of PDMS are oftentimesfragile. In one embodiment, the gas exchanger comprises a thick piece ofPDMS. However, thick pieces of PDMS are oftentimes absorbing. In oneembodiment, a gas exchanger can comprise a gas-impermeable substratewith gas-permeable regions, or pores. The gas-impermeable material maybe a rigid polymer. The gas-permeable material may be an elastomericpolymer. It is believed that gas-impermeable substrate withgas-permeable regions is itself a novel embodiment for use with anyfluidic device. Finally, another embodiment to solve the problem theelastomeric, high-absorbing, gas-permeable microfluidic device of U.S.Pat. No. 8,647,861 is to both encapsulate one or more channels of saidmicrofluidic device with said gas exchanger, and also put thin films ormasks of rigid polymer in contact with said outside portions of saidmicrofluidic device that are not the gas exchanger in order to limit gastransport from the ambient environment into the microfluidic device.

FIGS. 25A and 25B depict fluorescent images of fluidic layer assembliesof perfusion manifold assemblies, either comprising a combined gasketingand capping layer or separate, yet bonded low-absorbing capping andlow-absorbing gasketing layers. The fluorescent signal is given off bythe compound rhodamine, which was exposed to the system components.Bright white in the images indicate areas where compound has absorbed.FIG. 25A depicts the resulting fluorescence in a combined gasketing andcapping layer following exposure to the fluorescent small molecule,rhodamine, which is known to absorb. FIG. 25B depicts the resultingfluorescence in a fluidic layer assembly comprising separatelow-absorbing gasketing and low-absorbing capping layer.

FIG. 26 shows comprehensive images of all the results of fluidic layerassembly small molecule absorption for multiple embodiments. Anabsorbing perfusion manifold assembly was tested. A supposedlylow-absorbing perfusion manifold assembly was tested, comprising a COPcapping layer and a non-coated SEBS gasketing layer was tested. Fivelow-absorbing perfusion manifold assemblies, comprising a COP cappinglayer and a Parylene coated SEBS gasketing layer were also tested.Bright white in the images indicate areas where the fluorescent moleculehas absorbed.

FIGS. 27A and 27B show fluorescent molecule absorption in the resistors(27), having been capped with SEBS and COP respectively. Note that inFIG. 27B the bright white lines represent an optical artifact(reflection of light by the walls of the channel) as opposed to emissionof fluorescence.

FIGS. 28A and 28B show results from an experiment wherein liver cellswere seeded in a low-absorbing, gas-impermeable microfluidic devicefabricated from COP. FIG. 28A depicts liver cells in a low-absorbing,gas-impermeable microfluidic device fabricated from COP on day 7 ofculture. FIG. 28B shows comparable albumin production, a readout ofliver function, in the liver cells in both the low-absorbingmicrofluidic device and the absorbing microfluidic device.

FIGS. 29A and 29B depict depletion of the drug Diazepam, which is knownto absorb highly in PDMS, in both a plate and low-absorbing,gas-impermeable microfluidic device fabricated from COP. FIG. 29Adepicts, with a black solid line, an expected depletion model of thedrug Diazepam in a plate culture calculated from in vivo drug clearancedata (liver metabolism). The data points depict concentration decline ina plate experiment, with the dotted yellow line being a best-fit line tothe data. As would be expected, the decline is log-linear with respectto time, indicating metabolism as the primary driver for compound loss.The slope of this line indicates the rate of metabolism, or intrinsicclearance. Since the data has a lower slope than the model predicted, wecan see from the graph that the measure rate of metabolism in the platewas much lower than in vivo. FIG. 29B similarly depicts an expecteddepletion model of the drug Diazepam in an embodiment of a microfluidicdevice based on in vivo data from the literature. Data from actualcell-based experiments is shown for both a device fabricated from ahighly absorbing PDMS device and a low-absorbing microfluidic devicefabricated from COP. Best-fit lines are drawn through both data sets,with the slope indicating the rate of metabolism. As is readilyapparent, the COP microfluidic device (here designated as “NewLiver-Chip”) matches the in vivo predicted value much more closely thanthe PDMS device. However, the PDMS device appears to have a higher rateof metabolism based on the steeper slope. It is also important to notethat the PDMS Liver-Chip data is not well approximated by a line on thelog scale, as would be expected if metabolism was the only driver forcompound loss. Indeed, taken together (that is to say, knowing thatdiazepam absorbs into PDMS, seeing the poor fit of the data to ametabolism curve, and observing the higher than expected rate ofcompound loss), this clearly demonstrates an over-prediction ofmetabolism in the PDMS device and accurate prediction in thenon-absorbing system.

FIG. 30 shows the predicated clearance of Diazepam in vivo, theclearance measured on a plate, measured in an absorbing microfluidicdevice (12) fabricated from PDMS, and a low-absorbing, gas-impermeablemicrofluidic device (13) fabricated from COP. As can be seen in thegraph, the low-absorbing device, here termed “New Liver-Chip”, mostclosely matches the in vivo rate, and therefore is most predictive.

FIG. 31 depicts microscopy images of the Parylene-coated PDMS gasketsafter having been exposed to Rhodamine B. A slight pinkish hue isvisible, indicating some absorption is present on the corners of thegaskets, perhaps due to a poor coating on the edges.

FIG. 32 depicts microscopy images of the Parylene-coated PDMS gasketsafter having been exposed to Rhodamine B. A slight pinkish hue isvisible, indicating some minimal absorption is present. However, theabsorption is primarily localized to areas with sharp corners. Someabsorption can be seen inside of the via, but it was minimal anddifficult to visualize, and quite possibly an optical artifact unrelatedto absorption.

FIG. 33A-B shows the results of studies on absorption into Parylenecoated materials. FIG. 33A depicts the fraction of Coumarin recoveredfrom the solutions. FIG. 33B depicts the fraction of Rhodamine Brecovered from the solutions for coatings of varying thicknesses on twomaterials known to absorb. FIG. 33A shows that some Coumarin wasabsorbed by both the coated PDMS and SEBS with different coatingthicknesses. FIG. 33B shows that minimal Rhodamine B was absorbed by thePDMS and SEBS at the different coating thicknesses.

FIG. 34 depicts some different varieties of gas-exchangers, includingTeflon AF2400, TPX, and porous PET.

FIGS. 35A and 35B illustrate the difference in stretch between thecenter of the membrane and a section of the membrane close to the portsin a completely flexible absorbing microfluidic device that is stretchedvia vacuum application to the working channels. FIG. 35A demonstratesdeformation of the channel due to engagement with the perfusion manifoldassembly, even before stretching the membrane. FIG. 35B shows this samedevice under stretch. It can be seen that in the absorbing microfluidicdevice that is actuated in this manner, that there is a non-uniformstretch profile along the channel length, especially but not limited to,the area toward the edges of the working channels and far away from theworking channels.

FIG. 36 depicts the difference in stretch over the length of theabsorbing microfluidic device. In this embodiment of stretch, onlyapproximately 20% of the culture area is under the applied stretch basedon a preliminary study.

FIG. 37A-B display the membrane before and after the pressuredifferential is applied across the top and bottom channels. In someembodiments stretch is achieved by having a pressure differential acrossthe top channel and bottom channel, as to push the membrane in thedirection of the lower pressure channel.

FIG. 38 shows a side view of a 50 μm thick PDMS membrane, having hadfluorescent beads embedded in it, imaged on a confocal microscope atdifferent pressure differentials. The membrane deflects into the upperchamber of the device. The fluorescent membrane was fabricated by spincoating a layer of PDMS with fluorescent beads. It may be seen in FIG.38 that the greater the pressure differential the greater the level ofstretch of the membrane.

FIG. 39 shows a scatter plot for various levels of applied differentialpressure across a 50 μm thick PDMS membrane vs. measured strain, fitwith a curve to get the relationship between applied pressure andstrain. As expected, in the pressure regime tested, the relationship islinear.

FIG. 40 shows 20 μm thick PDMS membrane actuation resulting from apressure differential across the PDMS membrane imaged on a confocalmicroscope.

FIG. 41 shows a scatter plot for various levels of applied differentialpressure across a 20 μm thick PDMS membrane vs. measured strain, fitwith a curve to get the relationship between applied pressure andstrain. As expected, in the pressure regime tested, the relationship islinear.

FIG. 42 depicts strain from applied transmembrane pressure differentialsalong with model predictions and an indication of different stretchregimes based on the dominating physics.

FIG. 43 depicts strain from applied transmembrane pressure differentialin the mechanical advantage region/regime, which is the pressure rangewhere the pressure range that is most physiologically relevant (i.e.pressure seen in vivo).

FIG. 44 depicts the physiologically relevant pressures seen in thevasculature. Indeed, within the capillaries, which many Organ-Chips seekto emulate, the in vivo relevant pressure is between 2.5-4 kPa.

FIGS. 45A and 45B depict microfluidic devices for use in livervalidation experiments. FIG. 45A shows a gas-permeable, low-absorbingmicrofluidic device comprising an 11% porous PET and PDMS thin-film gasexchanger. FIG. 45B depicts a low-absorbing, gas-permeable microfluidicdevice comprising a PDMS thin-film gas exchanger.

FIGS. 46A, 46B, 46C, and 46D depict liver cell (hepatocyte) layermorphology in an absorbing microfluidic device fabricated from PDMS onsuccessive days. FIG. 46A shows the monolayer on Day 1. FIG. 46B showsthe monolayer on Day 3. FIG. 46C shows the monolayer on Day 6. FIG. 46Dshows the monolayer on Day 10. The monolayer appeared to be maintainedthrough Day 10, with slight morphological decline.

FIGS. 47A, 47B, 47C, and 47D depict the morphology of the cell monolayer(33) in a low-absorbing, gas-impermeable microfluidic device (13)constructed from COP. FIG. 47A shows the monolayer (33) on Day 1. FIG.47B shows the monolayer (33) on Day 3. FIG. 47C shows the monolayer (33)on Day 6. FIG. 47 D shows the monolayer (33) on Day 10. The monolayer(33) appeared to be declining rapidly over the course of the 10 days,with most cells completely dead or dying by Day 10.

FIGS. 48A, 48B, 48C, and 48D depict the morphology of the cell monolayer(33) in a low-absorbing, gas-permeable microfluidic device (1) with aporous PET and thin film PDMS gas exchanger (9). FIG. 48A shows themonolayer (33) on Day 1. FIG. 48B shows the monolayer (33) on Day 3.FIG. 48C shows the monolayer (33) on Day 6. FIG. 48D shows the monolayer(33) on Day 10. The monolayer (33) appeared to be maintained through Day10, with slight morphological decline (similar to the gas-permeable, butabsorbing device in FIG. 46A-D).

FIGS. 49A, 49B, 49C, and 49D depict the morphology of the cell monolayer(33) in a low-absorbing, gas-permeable microfluidic device (1) with athin film PDMS gas exchanger (9). FIG. 49A shows the monolayer (33) onDay 1. FIG. 49B shows the monolayer (33) on Day 3. FIG. 49C shows themonolayer (33) on Day 6. FIG. 49D shows the monolayer (33) on Day 10.The monolayer (33) appeared to be maintained through Day 10, with slightmorphological decline (similar to the gas-permeable, but absorbingdevice in FIG. 46A-D).

FIGS. 50A, 50B, 50C, and 50D depict the MRP2 signal of the BileCanaliculi of all the conditions at Day 14. FIG. 50A shows the BileCanaliculi MRP2 signal on an absorbing microfluidic device (12)constructed from PDMS on Day 14. FIG. 50B shows the Bile Canaliculi MRP2signal on a low-absorbing, gas-impermeable microfluidic device (13)constructed from COP on Day 14. FIG. 50C shows the Bile Canaliculi MRP2signal on a low-absorbing, gas-permeable microfluidic device (1) with aporous PET and thin film PDMS gas exchanger (9) on Day 14. FIG. 50Dshows the Bile Canaliculi MRP2 signal on a low-absorbing, gas-permeablemicrofluidic device (1) with a thin film PDMS gas exchanger (9) on Day14. There was no MRP2 signal for any of the conditions on Day 14.

FIGS. 51A and 51B depict average Albumin secretion in four differentmicrofluidic device conditions on Day 4, Day 9 and Day 13. Albuminsecretion is lower in both the low-absorbing, gas-permeable microfluidicdevice with a porous PET and thin film PDMS gas exchanger and thelow-absorbing, gas-permeable microfluidic device with a thin film PDMSgas exchanger than the absorbing microfluidic device constructed fromPDMS. However, there is a significant improvement from thelow-absorbing, gas-impermeable microfluidic device constructed from COP,which is gas-impermeable and non-absorbing.

FIG. 52 depicts the method of introducing an oxygen gradient into thelow-absorbing, gas-permeable microfluidic device comprising a gasexchanger, using said gas exchanger to selectively introduce a gas intothe microfluidic device from the vasculature channel only, whilecreating a diffusive barrier to the oxygen-rich ambient environment.

FIG. 53 depicts the morphology of the cell type Caco-2 in alow-absorbing, gas-permeable microfluidic device. This is an intestinecell line that could benefit from the creation of oxygen gradients fromthe vasculature channel into the apical channel, which represents theintestinal lumen.

FIG. 54 depicts the oxygen concentration profile of the low-absorbing,gas-permeable microfluidic device sampled at the four different ports:top channel inlet port, top channel outlet port, bottom channel inletport and bottom channel outlet port. In this experiment, oxygen-richmedia was perfused into both the apical and basal inlets. Because of thegas exchanger, the basal channel remained oxygenated, while the apicalchannel became nearly depleted of oxygen. This is a highly desirable andsought-after result, as this recapitulates the oxygen gradients seen inthe colon, which are necessary to imitate the in vivo condition.Specifically, this is important for maintaining adequate oxygen levelsto supply intestinal cells with needed levels to maintain homeostasis,while creating a low-oxygen environment in the channel representing thelumen, where anaerobic bacteria, such as Clostridium symbiosum, thrive.

FIGS. 55A, B, C and 56A, B, C show hepatocyte attachment and morphologyin both a low-absorbing, gas-impermeable microfluidic device fabricatedfrom COP and a high-absorbing, gas-permeable microfluidic devicefabricated from PDMS on day 1, day 2 and day 3 of cell layer growth.FIG. 55A shows hepatocyte attachment and morphology in a low-absorbing,gas-impermeable microfluidic device fabricated from COP on day 1. FIG.55B shows hepatocyte attachment and morphology in a low-absorbing,gas-impermeable microfluidic device fabricated from COP on day 2. FIG.55C shows hepatocyte attachment and morphology in a low-absorbing,gas-impermeable microfluidic device fabricated from COP on day 3. FIG.56A shows hepatocyte attachment and morphology in a high-absorbing,gas-permeable microfluidic device fabricated from PDMS on day 1. FIG.56B shows hepatocyte attachment and morphology in a high-absorbing,gas-permeable microfluidic device fabricated from PDMS on day 2. FIG.56C shows hepatocyte attachment and morphology in a high-absorbing,gas-permeable microfluidic device fabricated from PDMS on day 3.

FIGS. 57A and 57B show hepatocyte and LSEC morphologies on day 9 in ahigh-absorbing, gas-permeable microfluidic device fabricated from PDMS.FIG. 57A shows hepatocyte morphology on day 9 in a high-absorbingmicrofluidic device fabricated from PDMS. FIG. 57B shows LSEC morphologyon day 9 in a high-absorbing microfluidic device fabricated from PDMS.

FIGS. 58A and 58B show hepatocyte and LSEC morphologies on day 9 in alow-absorbing, gas-impermeable microfluidic device fabricated from COP.FIG. 58A shows hepatocyte morphology on day 9 in a low-absorbing,gas-impermeable microfluidic device fabricated from COP. FIG. 58B showsLSEC morphology on day 9 in a low-absorbing, gas-impermeablemicrofluidic device fabricated from COP. Both hepatocytes and LSECsshowed comparable morphologies and maintained monolayers in both thelow-absorbing, gas-impermeable microfluidic device and thehigh-absorbing, gas-permeable microfluidic device on day 9.

FIGS. 59A and 59B show bile canaliculi fluorescence staining via MRP2 atday 9 of cell layer culture on two different microfluidic devices. FIG.59A shows bile canaliculi fluorescence staining via MRP2 on ahigh-absorbing, gas-permeable microfluidic device fabricated from PDMSusing a 20× microscope objective on day 9 of cell layer culture. FIG.59B shows bile canaliculi fluorescence staining via MRP2 on ahigh-absorbing, gas-permeable microfluidic device fabricated from COPusing a 20× microscope objective on day 9 of cell layer culture.

FIG. 60 depicts an overview of albumin production across fourconditions. The microfluidic devices tested include: five low-absorbing,gas-impermeable microfluidic devices fabricated from COP with topchannel flow rates of 0 μL/hr and bottom channel flow rates of 300μL/hr; five low-absorbing, gas-impermeable microfluidic devicesfabricated from COP with top channel flow rates of 10 μL/hr and bottomchannel flow rates of 300 μL/hr; five absorbing, gas-permeablemicrofluidic devices fabricated from PDMS with top channel flow rates of10 μL/hr and bottom channel flow rates of 30 μL/hr; and fivehigh-absorbing, gas-permeable microfluidic devices fabricated from PDMSwith top channel flow rates of 10 μL/hr and bottom channel flow rates of300 μL/hr.

FIG. 61 shows CYP1A2 levels at day 14 following lysing of themicrofluidic devices shown in FIG. 60.

FIG. 62 shows CYP3A4 levels at day 14 following lysing of themicrofluidic devices shown in FIG. 60.

FIG. 63 shows CYP2A6 levels at day 14 following lysing of themicrofluidic devices shown in FIG. 60.

FIG. 64 shows an experimental matrix in which all the experimentalconditions may be seen for an optimization study aimed at sustainingLiver-Chip viability and function. The microfluidic devices comprised:three low-absorbing, gas-impermeable microfluidic devices fabricatedfrom COP with media equilibrated with 100% oxygen (i.e. 100 kPa, no CO2equilibration, with a 150 μL/hr flow rate in the top channel and a 150μL/hr flow rate in the bottom channel being run on a culture module;three low-absorbing, gas-impermeable microfluidic devices fabricatedfrom COP, with 21% oxygen media equilibration and 5% carbon dioxide, a150 μL/hr flow rate in the top channel and a 150 μL/hr flow rate in thebottom channel being run on a culture module; three low-absorbing,gas-impermeable microfluidic devices fabricated from COP, with mediaequilibrated to 21% oxygen and 5% carbon dioxide, a 150 μL/hr flow ratein the top channel and a 150 μL/hr flow rate in the bottom channel, andadditionally having 15 mM HEPES in the media to pH buffer the media,being run on a culture module; low-absorbing, gas-impermeablemicrofluidic devices fabricated from COP, with media equilibrated to 21%oxygen and 5% carbon dioxide, at a 300 μL/hr flow rate in the topchannel and a 300 μL/hr flow rate in the bottom channel being run on asyringe pump; two high-absorbing, gas-permeable microfluidic devicesfabricated from PDMS, with media equilibrated to 21% oxygen and 5%carbon dioxide, with a 300 μL/hr flow rate in the top channel and a 300μL/hr flow rate in the bottom channel being run on a syringe pump; andtwo high-absorbing, gas-permeable microfluidic devices, fabricated fromPDMS, with media equilibrated with 21% oxygen and 5% carbon dioxide,with a 30 μL/hr flow rate in the top channel and a 30 μL/hr flow rate inthe bottom channel being run on a culture module.

FIG. 65 depicts albumin production at each condition shown in FIG. 64.

FIGS. 66A, 66B and 66C show an experimental setup for reciprocation ofmedia. The setup involves pumping media through a low-absorbing,gas-impermeable microfluidic device fabricated from COP or ahigh-absorbing, gas-permeable microfluidic device using a syringe pump.The media collects in an external reservoir that is connected to theoutlet port. Because this reservoir is “open” to the externalenvironment, the media is able to equilibrate to the ambient oxygenconcentration in the air. If the cells in the device have depleted theoxygen in the media, oxygen will quickly diffuse into the media tore-saturate with dissolved oxygen. Once most of the media has beenpumped out of the syringe, the syringe pump reverses direction andbegins to pump media from the external reservoir back into the syringe.

FIG. 67 depicts the flow process of the experimental setup shown inFIGS. 66A, 66B and 66C, where the media is pushed back and forth throughthe microfluidic device from the syringe and external reservoir. In FIG.67, the media is first drawn from the external reservoir, through themicrofluidic device, into the syringe. The media is then optionally heldstatic in the syringe in the middle panel of the figure. The media isthen pushed out of the syringe, back through the microfluidic device,into the external reservoir. The external reservoir may alternatively beknown as a reservoir or fluid reservoir.

FIG. 68 shows the results of an experiment assessing the absorption ofParylene coated SEBS and Parylene coated E140 compared to the absorptionof known low-absorbing materials, such as glass and COP, and a controlsolution of the drug (Coumarin, which is known to absorb highly) not incontact with a material. Only non-coated materials were seen to absorb.

FIG. 69 depicts one embodiment of a low-absorbing, gas-permeablemicrofluidic device where the channel components are fabricated out ofCOP (which is known not to absorb), the gasketing material is fabricatedfrom PDMS with a Parylene coating (which the coating is known not toabsorb). Also pictured is one embodiment of a perfusion manifoldassembly microfluidic device carrier for the use of interfacing themicrofluidic device with a perfusion manifold assembly. This embodimentof the microfluidic device is compatible with the face-sealing gasketingmethod in one preferred embodiment of the device/perfusion manifoldassembly.

FIG. 70 shows the recovered concentration of Midazolam, a small moleculeknown to absorb, from a solution that had been in contact with variousmaterials, including glass, polypropylene, polystyrene, PDMS, SEBS andCOP.

FIG. 71 shows the recovered concentration of Bufuralol, a compound knownto absorb, from a solution that had been in contact with variousmaterials, including glass, polypropylene, polystyrene, PDMS, SEBS andCOP. Note that data is plotted for PDMS, but that the recoveredconcentrations were below the lower limit of detection (that is to saythe compound effectively completely absorbed into the material and wasremoved from the dosing solution).

FIG. 72 shows a computational model of Midazolam absorbing into ahigh-absorbing, gas-permeable microfluidic device fabricated from PDMS.FIG. 72 illustrates one of the challenges with absorption; even thoughboth the top and bottom channel were dosed with compound and even thoughflow rate (150 uL/hr) is higher than is typically run in thesemicrofluidic device (i.e. “best case scenario”) only the cells at thebeginning of the cell culture channel are contacted by the drug beforeit is absorbed into the PDMS. The latter half of the microfluidicdevices are exposed to a concentration of compound that is nearly “0”.

FIG. 73 shows an exemplary embodiment of a high-absorbing, gas-permeablemicrofluidic device in a microfluidic device holder or clip, such thatthe high-absorbing, gas-permeable microfluidic device may be fluidicallyconnected to a perfusion manifold assembly.

FIG. 74 shows an exemplary embodiment of a perfusion manifold assembly.

FIG. 75 shows the distribution of small-molecules and how likely theyare to absorb into surrounding materials. Approximately ˜40% ofsmall-molecules previously tested do not absorb. Approximately ˜40% ofsmall-molecules somewhat absorb. Approximately ˜20% of small-moleculeseffectively absorb completely on the time and length scales of anOrgan-Chip.

FIG. 76 shows a listing of compounds tested for absorption, theirmolecular weight (MW), one of their physicochemical parameters (log P),and the partition coefficient for the level of absorption into PDMS andthe material of the perfusion manifold assembly (pod).

FIGS. 77A, 77B, and 77C show a selection of the physical components ofthe compound distribution kit. In one embodiment, the physical componentof the compound distribution kit includes a plurality of microfluidicdevices comprising a poreless membrane, a plurality of perfusionmanifold assemblies, a plurality of filters, and a quick start guide.FIG. 77A shows three microfluidic devices in microfluidic device holdersor carriers and three open sterility bags, which had originallycontained the three microfluidic devices. FIG. 77B shows three perfusionmanifold assemblies in a sterile container. FIG. 77C shows two filtersin sterile packaging.

FIG. 78 shows an example of a calculator or absorption calculator. FIG.78 shows one embodiment where the calculator is a Microsoft Excelcalculator. The calculator is part of the digital component of thecompound distribution kit.

FIG. 79 shows one embodiment of a timeline for the compound distributionkit. The first step is to set up the culture module, which in oneembodiment is an Emulate Zoe™. Step two is to prepare dosing solution(s)and additional needs for calibration. Step three is to dose themicrofluidic devices (chips) and perfusion manifold assemblies (pods) atdesired time points. The fourth step is to quantify effluent sample(compound) concentration, for example with an LCMS. The fifth step is toassess cellular exposure compound concentrations. FIG. 79 also showsperfusion manifold assemblies preparing to be fluidically connected to aculture module, two tubes of solution, and examples of a calculator andgraphical calculator results.

FIG. 80 shows one embodiment of three perfusion manifold assembliespreparing to be fluidically connected to a culture module.

FIG. 81 shows three perfusion manifold assemblies with their lidsremoved. In FIG. 81 the perfusion manifold assemblies each have twoinlet and two outlet reservoirs and the two inlet reservoirs are shownfilled with a fluid.

FIG. 82 shows an exemplary embodiment of a culture module.

FIG. 83 shows two sets of dilutions acceptable for a five-pointcalibration.

FIG. 84 shows a Microsoft Excel calculator outputting absorption data aspart of the digital component of the compound distribution kit.

FIG. 85 shows a flow chart of preparing microfluidic devices andperfusion manifold assemblies for use with a culture module. FIG. 85first shows perfusion manifold assemblies and microfluidic devices (incarriers or holders) either in sterile packaging or recently removedfrom sterile packaging. FIG. 85 then shows the microfluidic devices inan orientation to be fluidically connected to the perfusion manifoldassemblies. FIG. 85 then shows the microfluidic devices fluidicallyconnected to the perfusion manifold assemblies and the inlet reservoirsof the perfusion manifold assemblies filled with fluid. Finally, FIG. 85shows perfusion manifold assemblies in an orientation to be fluidicallyconnected to a culture module.

FIGS. 86A and 86B show examples of compound distribution kit output forminimal absorption. FIG. 86A shows a graph of the outlet concentrationin one channel of a microfluidic device for a case of minimal exposure.FIG. 86B shows a graph of the cellular exposure range in one channel ofa microfluidic device for a case of minimal absorption, which uses thedata in FIG. 86A to compute a minimum and maximum possible concentrationof compound “seen” by the cells inside the microfluidic device.

FIGS. 87A and 87B show examples of compound distribution kit output fornearly complete absorption of a compound. FIG. 86A shows a graph of theoutlet concentration in one channel of a microfluidic device for a caseof nearly complete absorption. FIG. 86B shows a graph of the cellularexposure range in one channel of a microfluidic device for a case ofnearly complete absorption.

FIGS. 88A, 88B, 88C, and 88D show example calculator outputs for thecompound Rhodamine. FIG. 88A shows the outlet concentration of Rhodaminein a first channel of one or more microfluidic devices. FIG. 88B showsthe cellular exposure concentration range of Rhodamine in a firstchannel of one or more microfluidic devices. FIG. 88C shows the outletconcentration of Rhodamine in a second channel of one or moremicrofluidic devices. FIG. 88D shows the cellular exposure concentrationrange of Rhodamine in a second channel of one or more microfluidicdevices.

FIG. 89 shows the results from absorption testing microfluidic devicesand perfusion manifold assemblies without cells. The results can be usedto put error bars, or confidence intervals, on exposure concentrationsin actual drug studies with cells. Exposure concentration confidenceintervals decrease with experiment duration, as the recoveredconcentration rises, with tighter confidence intervals at latertimepoints.

FIGS. 90A and 90B show an example dose-response curve for Rhodamine fora compound distribution kit calculator. FIG. 90A shows a dose-responseconfidence interval chart for Rhodamine for a six-hour timepoint. FIG.90b shows a dose-response confidence interval chart for Rhodamine for a72-hour timepoint.

FIG. 91 shows a chart detailing recommended media collection time pointsgiven an experiment duration, which would be defined by the particularsof the compound study of interest.

FIG. 92 shows a COMSOL model that can predict the outlet concentrationsof compounds based on parameters obtained from static vial studies.COMSOL models can help inform flowrates and other experimentalparameters.

FIG. 93 shows a diagram of a “halo chip” or microfluidic device with thecapability of creating a desired gaseous environment within the channelsof the microfluidic device. The microfluidic device shown in FIG. 93 hasa gas channel that runs around the perimeter of the working or cellchannels of the microfluidic device. A gas, such as nitrogen or oxygen,may be flowed into the gas channels of the microfluidic device. The bodyof the microfluidic device comprises a permeable material, such as PDMS.The gas may transport through the body of the microfluidic device intothe working or cell channels of the microfluidic device. For example, ifan anaerobic environment is desired for the channels, nitrogen may beflowed through the gas channels. For another example, if a highlyoxygenated environment is desired for the channels, oxygen may be flowedthrough the gas channels. The microfluidic device shown in FIG. 93 mayalso comprise a check valve to allow the gas to leave the microfluidicdevice. Further, the microfluidic device in FIG. 93 may also comprisevacuum channels. When vacuum is applied to the vacuum channels themicrofluidic device may stretch to emulate cellular physiology in vivo.The microfluidic device in FIG. 93 may also comprise sensors, such asoxygen sensors, in order to monitor the gas levels within themicrofluidic device.

FIG. 94 shows a diagram of the fabricated “halo chip” or microfluidicdevice shown in the diagram of FIG. 93. The microfluidic device shown inFIG. 94 comprises gas channels in order to introduce a gaseousenvironment to the working or cell channels within the microfluidicdevice. A gas, such as oxygen, nitrogen, helium, carbon dioxide, amixture thereof, a smoke, a vapor, etc., may be introduced into the gaschannels of the microfluidic device. That gas may then diffuse throughthe body of the microfluidic device into the working or cell channels ofthe microfluidic device. Cell viability may be improved when the cellsare cultured in similar environments that they experience in vivo. Assuch, the ability to introduce in vivo relevant gas concentrations tothe cells within the microfluidic device allows scientists to achievebetter experimental results. The microfluidic device shown in FIG. 94may also comprise vacuum channels for stretching the microfluidicdevice, valves, sensors, channel inlets, channel outlets, etc.

FIG. 95 shows a comparison of computational (COMSOL) model flow studyresults and actual flow study results for the small-molecule compoundRhodamine. FIG. 95 shows that the flow results fit the COMSOL model forthe outlet concentrations of the compound. Rhodamine tends to have alower rate of absorption, but higher extent of absorption, which resultsin it saturating its surroundings over time. The importance of this isthat despite initially seeing huge losses of rhodamine, after a periodof time, the rate of rhodamine loss diminishes significantly.

FIGS. 96A and 96B show a comparison between computational (COMSOL) modelresults and actual experimental results for cellular exposure ranges ofthe small-molecule compound Rhodamine. FIG. 96A shows experimentalresults of the cellular exposure range of the small-molecule compoundRhodamine for a first channel of a microfluidic device. FIG. 96B showscomputational (COMSOL) model results of the cellular exposure range ofthe small-molecule compound Rhodamine for a single channel of amicrofluidic device. The charts in FIGS. 96A and 968 show that thecomputational (COMSOL) model accurately predicted Rhodamine absorptioninto the materials making up microfluidic devices, particularly PDMS.

FIGS. 97A and 97B show a comparison between computational (COMSOL) modelresults and actual experimental results for cellular exposure ranges ofthe small-molecule compound Rhodamine. FIG. 97A shows experimentalresults of the cellular exposure range of the small-molecule compoundRhodamine for a second channel of a microfluidic device. FIG. 97B showscomputational (COMSOL) model results of the cellular exposure range ofthe small-molecule compound Rhodamine for a second channel of amicrofluidic device. The charts in FIGS. 97A and 97B show that thecomputational (COMSOL) model accurately predicts small-moleculeabsorption into the materials making up microfluidic devices,particularly PDMS.

FIGS. 98A and 98B show a comparison between a computational (COMSOL)model results and actual experimental results for cellular exposureranges of the small-molecule compound Coumarin. FIG. 98A showsexperimental results of the cellular exposure range of thesmall-molecule compound Coumarin for a first channel of a microfluidicdevice. FIG. 98B shows computational (COMSOL) model results of thecellular exposure range of the small-molecule compound Coumarin for afirst channel of a microfluidic device. It was found that thecomputational (COMSOL) model did not accurately predict the absorption,because the model did not take into account the rest of the flow systemoutside the microfluidic device. For this experiment the microfluidicdevice was in fluidic communication with a perfusion manifold assembly.The compound Coumarin was especially susceptible to absorption into oneof the materials making up the perfusion manifold assembly, SEBS. Assuch, the computational (COMSOL) model did not accurately predict theabsorption into the entire flow system.

FIGS. 99A and 99B show a comparison between a computational (COMSOL)model results and actual experimental results for cellular exposureranges of the small-molecule compound Coumarin. FIG. 99A showsexperimental results of the cellular exposure range of thesmall-molecule compound Coumarin for a second channel of a microfluidicdevice. FIG. 99B shows computational (COMSOL) model results of thecellular exposure range of the small-molecule compound Coumarin for asecond channel of a microfluidic device. It was found that thecomputational (COMSOL) model did not accurately predict the absorption,because the model did not take into account the rest of the flow systemoutside the microfluidic device. For this experiment the microfluidicdevice was in fluidic communication with a perfusion manifold assembly.The compound Coumarin was especially susceptible to absorption into oneof the materials making up the perfusion manifold assembly, SEBS. Assuch, the computational (COMSOL) model did not accurately predict theabsorption into the entire flow system.

FIG. 100 shows experimental results for cellular exposure of thesmall-molecule compound Rhodamine in a two-channel microfluidic devicecomprising a PDMS membrane at a flow rate of 60 uL/hr.

FIG. 101 shows experimental results for cellular exposure of thesmall-molecule compound Rhodamine in a two-channel microfluidic devicecomprising a PDMS membrane without pores at a flow rate of 60 uL/hr.

FIG. 102 shows experimental results for cellular exposure of thesmall-molecule compound Coumarin in a two-channel microfluidic devicecomprising a PDMS membrane at a flow rate of 150 uL/hr.

FIG. 103 shows experimental results for cellular exposure of thesmall-molecule compound Coumarin in a two-channel microfluidic devicecomprising a PDMS membrane without pores.

FIG. 104 shows the unilateral or unidirectional flow of gas, in thiscase oxygen, through the gas exchanger into the body of thelow-absorbing, gas-permeable microfluidic device at a flow rate of 60uL/hr.

FIG. 105 shows a timeline for a flow test of two small-moleculecompounds, Drug X and Drug Y. The dose concentration of Drug X was 10 μMand the dose concentration of Drug Y was 1 μM. For the experiment shownin FIG. 105 the end point analysis was liquid chromatography-massspectrometry.

FIGS. 106A and 106B show a summary of flow studies of Drug X in a firstchannel of a two-channel microfluidic device. FIG. 106A shows the outletconcentration of Drug X over time. FIG. 106B shows cellular exposureranges in the first channel. FIGS. 106A and 106B show that Drug X wasabsorbed into the system. The loss of Drug X is consistent with a lowerabsorbing molecule as nearly all of the compound is recoverable at 72hours, showing that the microfluidic device material became saturated.FIGS. 106A and 106B show that over time cell exposure to Drug X wouldreach between 80-100%. The media carrying Drug X in FIGS. 106A and 106Balso contained 2% fetal bovine serum (FBS).

FIGS. 107A and 107B show a summary of flow studies of Drug X in a secondchannel of a two-channel microfluidic device. FIG. 107A shows the outletconcentration of Drug X over time. FIG. 107B shows cellular exposureranges in the first channel. FIGS. 107A and 107B show that Drug X wascompletely absorbed into the system. The second channel flow rate maypossibly be increased in order to lessen the amount of compoundabsorption.

FIGS. 108A and 1088 summarize flow studies of Drug Y in the firstchannel of a microfluidic device. FIG. 108A shows the outletconcentration of Drug Y over time. FIG. 108B shows the range of cellularexposure in the first channel of the microfluidic device over time. Thecompound loss is consistent with a moderately absorbing molecule asnearly all of the compound is recovered over 72 hours in the effluent,as the material making up the microfluidic device becomes saturated.Over time cellular exposure of Drug Y would be between 80-100%. Themedia carrying Drug Y in FIGS. 108A and 108B also contained 2% fetalbovine serum (FBS).

FIGS. 109A and 109B summarize flow studies of Drug Y in the secondchannel of a microfluidic device. FIG. 109A shows the outletconcentration of Drug Y over time. FIG. 109B shows the range of cellularexposure in the second channel of the microfluidic device over time. Thecompound loss in the second channel of the microfluidic device pointstowards absorption. The flow rate may be increased to perhaps decreasecompound absorption.

FIG. 110 shows multiple embodiments of a gas exchanger. In theembodiments shown, a substrate comprises regions which are filled byanother material. The regions may be pores. The pores may be entirely orpartially filled. Further, the pores may be filled as well as covered.The pores may be covered on one or both sides.

FIGS. 111A-G show multiple embodiments of recirculation methods betweentwo reservoirs, in the figures being an “in” reservoir and an “out”reservoir. FIGS. 111A-G demonstrate the effectiveness of silicon valves.FIG. 111A shows an embodiment of a recirculation setup using an umbrellavalve. FIG. 111B shows an embodiment of a recirculation setup using aduck-billed valve. FIG. 111C-E show multiple embodiments ofrecirculation setups using tubes and duck billed valves. FIG. 111F showsan embodiment of a recirculation setup using a tube and a duck-billedvalve. FIG. 111G shows an embodiment of a recirculation setup using atube and an umbrella valve.

FIG. 112 shows a graph of albumin production in a PDMS and COPmicrofluidic devices comprising liver cells before and afterreciprocating fluid. It may be seen in FIG. 112 that reciprocating fluidleads to an increase in albumin production as compared to single passflow.

FIG. 113 shows albumin production in PDMS microfluidic devicescomprising liver cells before and after reciprocating fluid. It may beseen that reciprocating fluid leads to an increase in albuminproduction.

FIG. 114 comprises an embodiment of a low-absorbing microfluidic devicecomprising a rigid body having a main channel, an elastomeric membranepositioned in that channel, working or gas channels, and elastomericwalls between said main channel and said working or gas channels.Alternatively, FIG. 114 shows a microfluidic device comprising alow-absorbing body having a main channel, a flexible membrane, workingor gas channels, and flexible walls between said main channel and saidworking or gas channels. Alternatively, FIG. 114 shows a microfluidicdevice comprising a substantially rigid body having a channel, saidchannel comprising a flexible membrane, wherein said membrane may bestretched by working or gas channels separated from said channel by oneor more flexible walls.

FIGS. 115A-D show the results of an experiment testing the absorption ofa compound, herein called Compound Z, in a PDMS microfluidic devicecomprising liver cells using the Compound Distribution Kit. FIG. 115Ashows nearly complete absorption of Compound Z at low flow rates, suchas 30 uL/hr. FIG. 115B shows that significant absorption (nearly 80%loss) of Compound Z at high flow rates, such as 150 uL/hr. FIG. 115Cshows cellular exposure of Compound Z in said first channel of thecompound at 30 uL/hr. FIG. 115D shows cellular exposure of Compound Z insaid first channel of the compound at 150 uL/hr. Experiments were alsorun at a higher concentration to compensate for compound loss. Increaseddosing concentration of Compound Z was conducted and the recoveredoutlet concentration was used as the effective “cellular exposureconcentration.” Increasing the dosing concentration increases thelikelihood of a false positive (compound is not toxic, but a toxiceffect is seen in the microfluidic device), but eliminates thepossibility of a false negative (compound is actually toxic, but themicrofluidic device does not show any toxic response). FIG. 116 shows adiagram of oxygen tensions in various human organs. Oxygen, carbondioxide, and various gases are known to influence the biologicalfunction of cells and can have a profound effect in tissues and variousdisease states. For example, oxygen tension differs dramatically in thehuman body across organs, yet traditional cell culture techniques do nottake this into account.

FIG. 117 shows a diagram of gas exchange in a PDMS microfluidic device.Per FIG. 117, the method of gas transport in the microfluidic deviceincludes gas exchange between an incubator and the microfluidic devicematerial, the microfluidic device material and the cell culture media,and the cell culture media and the cells.

FIG. 118 shows a diagram of the results of microfluidic device responseto various oxygen phases while in a cell culture incubator. Oxygenmeasurements were taken of a microfluidic device outlet under flow at 30μL/hr flow in a culture module, wherein the flow is with 18.5% oxygeninto the inlet. As seen in FIG. 118 the incubator starts at atmosphericoxygen levels (18.5% in a humidified incubator), reaches 1% oxygensetpoint (seen with a long tail-end), and returns to atmospheric oxygenupon the incubator being opened to the atmosphere.

FIG. 119 shows a diagram of the results experimental oxygen measurementsof microfluidic device outlets under water flow at 100 μL/hr in aculture module with either 18.5% oxygen (oxygenated), or 1-5% oxygen(hypoxic) concentrations, in a 1% oxygen incubator. The microfluidicdevice and system were equilibrated to the incubator environment for 12hours prior.

FIG. 120 shows a diagram of the results of a COMSOL Multiphysicssimulation plot of a PDMS microfluidic device first channel and secondchannel volume averages of the same conditions with oxygenated media.

FIG. 121 shows a diagram of results of a COMSOL Multiphysics simulationplot of PDMS microfluidic device first and second channel volumeaverages for 30 μL/hr and 1000 μL/hr flow rates with oxygenated inletwater in a 1% oxygen incubator.

FIG. 122 shows a diagram of results of recovery time when opening anincubator door. Oxygen measurements were taken at the outlet of amicrofluidic device under 100 μL/hr water flow in a culture moduleinside an incubator set to 1% oxygen. The microfluidic device, culturemodule, and remainder of system were equilibrated to the incubatorenvironment for 12 hours prior. The incubator door was opened for fiveseconds before starting measurements.

FIG. 123 shows a diagram of results of a COMSOL Multiphysics simulationplot of PDMS microfluidic device first and second channel volumeaverages of a static PDMS microfluidic device equilibrated to 1% oxygenand exposed to atmospheric oxygen.

FIG. 124 shows a diagram of results of a COMSOL Multiphysics simulationplot of PDMS microfluidic device first and second channel volumeaverages of a microfluidic device with seeded Caco-2 cells in cultureconditions or 18.5% oxygen incubator and 18.5% oxygen inlet water at 100μL/hr water flow rate.

FIG. 125 shows a diagram of a PDMS microfluidic device oxygenmicroenvironment with the addition of Caco-2 cells. FIG. 125 shows across-sectional surface pot of water oxygen concentrations in the centerof the microfluidic device.

FIG. 126 shows a diagram of one embodiment of a gas-exchangemicrofluidic device, comprising a gas-exchange channel used to introducegas into the body of the microfluidic device. The embodiment in FIG. 126comprises a body having a culture channel, a gas-exchange channel, and agas exchanger between said culture channel and said gas-exchangechannel. The embodiment in FIG. 126 is much like the device in FIG. 3,but also comprises a gas-exchange channel in contact with the gasexchanger in order to exchange a gas of a desired concentration with thechannels of the microfluidic device.

DESCRIPTION OF THE INVENTION

Several embodiments to improve compound distribution and absorbencywithin microfluidic devices are presented herein.

One exemplary embodiment of the present invention is a low-absorbingmicrofluidic device to conduct experiments, cellular and otherwise.Another exemplary embodiment of the present invention is a low-absorbingperfusion manifold assembly representing fluidic infrastructure aroundthe microfluidic device. Both the low-absorbing microfluidic device andthe low absorbing perfusion manifold assembly aim to minimize smallmolecule absorption, while allowing ambient gases to access experimentalregions of the devices, such as microfluidic channels.

U.S. Pat. No. 8,647,861 describes a microfluidic device, ororganomimetic device, or microfluidic device for the use of mimickingorgan function, comprising: a body having a central microchanneltherein; and an at least a partially porous membrane positioned withinthe central microchannel and along a plane, the membrane configured toseparate the central microchannel to form a first central microchanneland a second central microchannel, wherein a first fluid is appliedthrough the first central microchannel and a second fluid is appliedthrough the second central microchannel, the membrane coated with atleast one attachment molecule that supports adhesion of a plurality ofliving cells wherein the porous membrane is at least partially flexible,the device further comprising: a first operating channel separated thefirst and second central microchannels by a first microchannel wall,wherein the membrane is fixed to the first chamber microchannel wall;and wherein applying a pressure to the first operating channel causesthe membrane to flex in a first desired direction to expand or contractalong the plane within the first and second central microchannels. Manyembodiments of the present invention may be considered improvements onthe microfluidic device presented in U.S. Pat. No. 8,647,861, followingthe surprising discovery that the materials most commonly used tofabricate the microfluidic devices in U.S. Pat. No. 8,647,861 areabsorptive. In the process of fabricating a low-absorbing microfluidicdevice, both gas-impermeable and a gas-permeable option were designedand fabricated.

In some instances, such as when anaerobic bacteria are being cultured, amicrofluidic device fabricated from highly permeable materials may notbe desired. As such, one embodiment of the present invention is to maskthe microfluidic device with films of non-permeable materials.

One embodiment contemplated to control gas is a microfluidic devicecomprising one or more gas-exchange channels to flow a fluid, either agas or liquid, and exchange gas between a gas source and another one ormore channels within a microfluidic device. The gas-control microfluidicdevice allows the gas concentration within a gas-permeable microfluidicdevice to be controllable. A gas, such as oxygen, nitrogen, helium,carbon dioxide, a mixture thereof, a smoke, a vapor, etc., may beintroduced into the gas channels of the microfluidic device. The body ofthe microfluidic device comprises a permeable material, such as PDMS.The gas may transport through the body of the microfluidic device intothe working or cell channels of the microfluidic device. Cell viabilitymay be improved when the cells are cultured in similar environments thatthey experience in vivo. As such, the ability to introduce in vivorelevant gas concentrations to the cells within the microfluidic deviceallows scientists to achieve better experimental results. For example,if an anaerobic environment is desired for the channels, nitrogen may beflowed through the gas channels. For another example, if a highlyoxygenated environment is desired for the channels, oxygen may be flowedthrough the gas channels.

In one embodiment, the gas-exchange channel may be used in conjunctionwith a gas exchanger. In one embodiment, a microfluidic device iscontemplated comprising a body having a culture channel, a gas-exchangechannel, and a gas exchanger between said culture channel and saidgas-exchange channel, as shown in FIG. 126. The embodiment in FIG. 126is much like the device in FIG. 3, but also comprises a gas-exchangechannel (45) in contact with the gas exchanger (9) in order to exchangea gas of a desired concentration with the channels (3, 4) of themicrofluidic device.

Another embodiment contemplated to control gas is a “halo chip,” amicrofluidic device with the capability of creating a desired gaseousenvironment within the channels of the microfluidic device, as shown inFIGS. 93 and 94. The “halo chip” or gas control microfluidic device hasa gas channel that runs around the perimeter of the working or cellchannels of the microfluidic device. FIG. 93 shows a diagram of a “halochip” or microfluidic device (47) with the capability of creating adesired gaseous environment within the channels of the microfluidicdevice. The microfluidic device shown in FIG. 93 has a gas channel (45)that runs around the perimeter of the working or cell channels (3, 4) ofthe microfluidic device. A gas, such as nitrogen or oxygen, may beflowed into the gas channels of the microfluidic device. The body of themicrofluidic device comprises a permeable material, such as PDMS. Thegas may transport through the body of the microfluidic device into theworking or cell channels (3, 4) of the microfluidic device (47). Forexample, if an anaerobic environment is desired for the channels (3, 4),nitrogen may be flowed through the gas channels (45). For anotherexample, if a highly oxygenated environment is desired for the channels,oxygen may be flowed through the gas channels. The microfluidic device(47) shown in FIG. 93 may also comprise a check valve (46) to allow thegas to leave the microfluidic device. Further, the microfluidic device(47) in FIG. 93 may also comprise vacuum channels. When vacuum isapplied to the vacuum channels the microfluidic device (47) may stretchto emulate cellular physiology in vivo. The microfluidic device in FIG.93 may also comprise sensors, such as oxygen sensors, in order tomonitor the gas levels within the microfluidic device.

FIG. 94 shows different view of the “halo chip” or microfluidic device(47) shown in FIG. 93. The microfluidic device (47) shown in FIG. 94comprises gas channels (45) in order to introduce a gaseous environmentto the working or cell channels (3, 4) within the microfluidic device. Agas, such as oxygen, nitrogen, helium, carbon dioxide, a mixturethereof, a smoke, a vapor, etc., may be introduced into the gas channels(45) of the microfluidic device (47). That gas may then diffuse throughthe body of the microfluidic device into the working or cell channels(3, 4) of the microfluidic device (47). Cell viability may be improvedwhen the cells are cultured in similar environments that they experiencein vivo. As such, the ability to introduce in vivo relevant gasconcentrations to the cells within the microfluidic device allowsscientists to achieve better experimental results. The microfluidicdevice (47) shown in FIG. 94 may also comprise vacuum channels forstretching the microfluidic device, valves, sensors, channel inlets,channel outlets, etc.

In some instances, especially those involving small molecule agents,absorbency into PDMS is problematic. One of the first iterations of theinvention presented herein in order to overcome said absorbency is agas-impermeable, low-absorbing microfluidic device. The gas-impermeablemicrofluidic device comprising: a body having at least one channeltherein, and a membrane positioned in that channel. The gas-impermeablemicrofluidic device comprising: a body having a central microchanneltherein; and an at least partially porous membrane positioned within thecentral microchannel and along a plane, the membrane configured toseparate the central microchannel to form a first central microchannel,or bottom channel, and a second central microchannel, or topmicrochannel, wherein a first fluid is applied through the first centralmicrochannel and a second fluid is applied through the second centralmicrochannel. FIG. 5 depicts an embodiment of a microfluidic deviceentirely fabricated out of gas-impermeable materials, such as COP andSEBS gasketing layers. The gas-impermeable microfluidic device (13) hasa body fabricated out of COP in order to be low-drug absorbing. FIG. 6depicts the same embodiment of a microfluidic device fabricated out ofentirely gas-impermeable materials exploded as to see the differentlayers. The gas-impermeable microfluidic device may include similarlayers as the absorbent microfluidic device (12) above mentioned or thelow-absorbent microfluidic device (1) presented herein. These elementsinclude, but are not limited to, the top channel layer (6) comprising atop channel (3), the bottom channel layer (8) comprising a bottomchannel (4), and a membrane (7) between the top channel layer (6) andthe bottom channel layer (8). The embodiment depicted in FIG. 6 containstwo gaskets (5) instead of one gasket (5) covering the entire topsurface of the microfluidic device (13) as depicted in FIG. 1. Theformat of the gas-impermeable microfluidic device (13) is compatiblewith the infrastructure of the absorbent microfluidic device (12)described in U.S. Pat. No. 8,647,861. The embodiment of thegas-impermeable microfluidic device (13) in FIG. 5 is more amenable tolarge scale manufacturing than the absorbent microfluidic device (12)described in U.S. Pat. No. 8,647,861, the reason being that thegas-impermeable microfluidic device is amenable to thermoplasticinjection molding processes. Notably missing from this design areworking channels, as microfluidic device fabricated from rigid materialscannot be stretched using working channels, as the culture channel wallsare also rigid. If the membrane is elastomeric, then differentialstretching is a possibility. The latter embodiment is discussed infurther detail later.

In some experimental pursuits stretching of the microfluidic deviceusing is advantageous. The microfluidic device fabricated from entirelyrigid materials was modified to allow the membrane to be stretchedthrough working channels. An embodiment of the low-absorbingmicrofluidic device was fabricated in order to include working or gaschannels, and have the membrane be able to be stretched with saidworking or gas channels. FIG. 114 shows one embodiment of thislow-absorbing microfluidic device (49) comprising an elastomericmembrane (7) and elastomeric channel walls (48). The low-absorbingmicrofluidic device (49) may be predominantly rigid, while having a mainchannel comprising elastomeric walls and an elastomeric membrane (7).The main channel may comprise a first channel (3) and a second channel(4). The membrane (7) may be elastomeric to facilitate gas transport oneither side of said membrane. The walls of the channel (48) may beelastomeric to facilitate stretching of the membrane (7) if desiredthrough the use of gas or working channels (32). However, in someembodiments differential pressure may be used to stretch said membrane(7), and in that case the body and channel walls may be rigid, whilesimply the membrane (7) is elastomeric. In the embodiment where solelythe membrane is elastomeric, the amount of absorbing material may beminimized as the membrane may represent a small volume of the membranein one embodiment. In one embodiment, the microfluidic device comprisesa body having at least one channel (3, 4) therein, said channel havingelastomeric walls (48) and an elastomeric membrane (7), wherein at leasta portion of said body is rigid. Furthermore, the embodiment comprisingelastomeric channel walls (48) and a membrane (7) may necessitatefurther fabrication steps than an embodiment wherein the body isentirely rigid. In one embodiment, the microfluidic device comprises abody having at least one channel (3, 4) therein, said channel havingrigid walls and an elastomeric membrane (7), wherein at least a portionof said body is rigid. However, a substantially rigid microfluidicdevice fabricated with elastomeric channel walls requires furtherfabricate steps, and lamination fabrication would not be able to be aseffectively used.

As previously stated, the microfluidic device fabricated out of entirelyrigid materials may be modified to have an elastomeric membrane in orderto facilitate differential stretching. Differential stretching is shownin FIGS. 37A and 37B. The microfluidic device shown in FIGS. 37A and 37Bmay have a body (6, 8) of any material as long as the membrane (7) iselastomeric.

In some cases, these entirely gas-impermeable microfluidic devices causedeath of specimens, such as cells, as they are unable to access ambientgases, such as oxygen, which are required for essential biologicalfunctions, like respiration.

In order to overcome low oxygen levels in microfluidic devices, madeboth from rigid and elastomeric materials, several new techniques werecontemplated and then employed. One embodiment to overcome thegas-impermeability was to add supplements, such as hemoglobin, to themedia or fluid, such as to augment (e.g. increase) the gas carryingcapacity of the media or fluid. It was found, however, that thesesupplements are sometimes difficult to work with.

Another embodiment to overcome the gas-impermeability was to flow fluidsor media at high flow rates in order to introduce a higher concentrationof dissolved oxygen into the channels of the microfluidic device.Unfortunately, there are some disadvantages to high flow rates includingfluid or media waste. In the cases that cells are cultured in themicrofluidic device, important cellular signals can be washed away.Further, higher flow rates result in higher levels of shear which maynot always be favorable.

In order to overcome these disadvantages, fluid or media may berecirculated. Recirculation involves circulating substantially the samemedia through a microfluidic device at least twice. The media may beoxygenated between each circulation. Further, some experiments requirehigh-shear. For example, vascular cells may need to be exposed tohigh-shear flow in some experiments. High-shear applications require“rapid-recirculation,” and therefore large volumes of fluid. FIGS.111A-G show several embodiments proposed for recirculating media througha microfluidic device, such as that depicted in FIG. 5, using aperfusion manifold assembly, such as that depicted in FIG. 7. FIGS.111A-G show multiple embodiments of recirculation methods between tworeservoirs, in the figures being an “in” reservoir and an “out”reservoir. The general technique contemplated is to have valves andtubes connecting an inlet reservoir to an outlet reservoir, such asthose (19) in FIG. 7. Two reservoirs, separated by a wall are shown inFIGS. 111A-G. These reservoirs may be the reservoirs (19) in theperfusion manifold assembly (14). The reservoirs have fluid/liquid/mediain them. FIG. 111A shows an embodiment of a recirculation setup using anumbrella valve (50). During flow through the microfluidic device, thevalve remains closed and the “OUT” fills with fluid via flow through themicrofluidic device. This is a discontinuous, albeit rapid, refilling ofthe inlet reservoir with media from the “OUT” reservoir, through a holeor channel between the reservoirs, that is normally blocked by theone-way (check) valve It is contemplated that a larger check valve, suchas a umbrella valve (50), may be used during recirculation, as smallvalves are known to leak. FIG. 1118 shows an embodiment of arecirculation setup using a duck-billed valve (51). FIG. 111C-E showmultiple embodiments of recirculation setups using tubes (53) and duckbilled valves (51). FIG. 111F shows an embodiment of a recirculationsetup using a tube (53) and a duck-billed valve (51). The recirculationsetup shown in FIG. 111F was tested and showed favorable compatibilityand success with the culture module (82) and perfusion manifoldassemblies (14). FIG. 111G shows an embodiment of a recirculation setupusing a tube (53) and an umbrella valve (50). FIGS. 111A-G demonstratethe effectiveness of silicon valves. As well, it was contemplated to uselower resistance resistors to enable higher flow rates and lower shearif desired. Recirculation may be achieved using a mini-valve in avestigial channel of a perfusion manifold assembly. Recirculation mayalso be achieved using discontinuous application of pressure to outletsto “burst” the valve leading to recirculation. When the valve “bursts”it allows fluid from the outlet reservoir into the inlet reservoir.

Potential use cases for recirculation include physiologically-relevantcapillary-gel shear rates, neutrophil recruitment, with low perfusionmanifold assembly shear, but high microfluidic device shear, andthrombosis recapitulation in a microfluidic device, with low perfusionmanifold assembly shear, but high shear in the microfluidic device.

Sometimes, though, recirculation setups can be bulky and requireequipment that is difficult to use. In those cases, fluid may bereciprocated, or flowed back and forth through the device. Reciprocationis non-obvious in the case of studying cells in vitro as fluid in vivodoes not flow two ways. A surprising discovery was that cells in vitrodisplayed high levels of viability and organ-specific function withreciprocated media. Reciprocation can also be performed on microfluidicdevices in the culture module (42), as seen in FIG. 82, and has beentested as part of experiments to evaluate the rate of metabolism ofliver cells to low clearance compounds on the culture module (42). Inthe experiment, a “low volume” (200 uL) was rapidly reciprocated “backand forth” through the microfluidic device, in order to maximize contacttime between media-containing-compound and the cell layer in themicrofluidic device. This was achievable for more than 24 hours in amicrofluidic device without cells.

Different cell types may require different amounts of oxygen in order tothrive. If cellular health is a goal/requirement, the rate of oxygenentering the microfluidic device should be greater than oxygen uptakerate within the microfluidic device in order to ensure that cells haveaccess to as much oxygen as they require. For example, liver hepatocytesmay require atmospheric levels of oxygen, whereas some bacteria culturesin the gut may require very low oxygen concentrations, with atmosphericlevels being toxic. As such, microfluidic devices, especially those withapplications in cellular biology, would benefit by being low-absorbing,while still allowing necessary levels of oxygen to reach cells,experiments, etc. inside the microfluidic device. Oftentimes however,low-absorbing materials tend to be gas-impermeable. In this way, amicrofluidic device minimizing the amount of material absorbency may bedesigned with a combination of gas-permeable and gas-impermeablecomponents to simultaneously minimize absorption and supply required gasto the cell layer.

An application for using microfluidic devices for Organ-Chips isunderstanding the resulting metabolite produced when cells are incontact with candidate compounds. In order to deduce intrinsic clearanceof drugs, for example, the metabolism or loss of the parent compoundoftentimes will need to be quantified. A first challenge in quantifyingmetabolism is if the metabolism is low. Low rates of metabolism can makeit difficult to detect loss of the parent compound, even if themicrofluidic device is non-absorbing. A practical limit of detection inan LC/MS instrument is ±25%. As such, a decrease in the concentration ofthe parent compound needs to be around 25% in order to detect/quantifymetabolism with confidence. Another challenge in quantifying metabolismis material absorption of the parent compound. If absorption into thematerial, such as PDMS, is significant, then the observed apparent rateof metabolism (if all of compound loss is attributed to metabolism) willover-estimate actual cell-mediated metabolism as the decrease incompound concentration will be incorrectly attributed to metabolism. Insome cases, all of the parent compound could be depleted by thematerial. In this case, absorption will prevent even an estimation ofthe upper possible rate of metabolism, since there will be no data toanalyze as all of the compound has been lost. Material absorption can becomputationally modeled and accounted for given information on thematerial-compound properties, like the rate and extent of absorption inthe material, experimental parameters, like dosing concentration andflow rate, and microfluidic device geometry as long as all of the parentcompound is not being depleted by the material. This however, requiresextensive studies to characterize the compound-material interaction aswell as computationally expensive models of the system to “subtract out”the contribution of material absorption to loss or disappearance ofcompound. To reiterate, though, if compound loss is complete, thesemodels cannot account for the contribution of absorption, as compoundloss is complete.

For example, quantifying the metabolism of Diazepam and amitriptyline inany system is difficult. Both Diazepam and amitriptyline are lowclearance compounds, meaning that they are slowly metabolized by theliver. A first challenge is that both Diazepam and amitriptylineoftentimes need long exposure times in microfluidic devices, such as aLiver-Chip. Long exposure times are needed in order to see appreciablecompound depletion in order to quantify metabolism. Long exposure timesoftentimes mean that very little media volume is provided to the cells,which also provides nutrients and carries away waste. If medianutrients, such as carbon components and dissolved oxygen, are depletedand waste is not sufficiently removed, cells may be damaged or even die.A second challenge is that both Diazepam and amitriptyline absorb intoPDMS, a common microfluidic device fabrication material. Long exposuretimes also mean that the drugs are in contact longer with the PDMS,which exacerbates compound loss due to absorption. PDMS absorption ofthe compounds can mask quantification of metabolism. FIGS. 15A and 15Bdepict the seriousness of Diazepam absorption into PDMS. As seen in FIG.15A, when media containing a compound is exposed to a sample of PDMSmaterial, which comprises the high-absorbing, gas-permeable microfluidicdevice (12) the decrease in compound concentration is significant inmagnitude and speed. Within 12 hours of exposure, nearly theconcentration of Diazepam has decreased by nearly ⅔. To contrast, it maybe seen in FIG. 15B that none of the dosing concentration of Diazepamwas lost to the bulk material of the low-absorbing, gas-impermeablemicrofluidic device (13) fabricated from COP. The experiments emphasizethe large absorbance difference between PDMS and COP. Experiments usingDiazepam were also run in microfluidic devices. FIG. 29A depicts anexpected depletion model of the drug Diazepam in a plate culturecalculated from in vivo drug clearance data versus actual data collectedfrom a plate culture. FIG. 29B depicts an expected depletion model ofthe drug Diazepam in a microfluidic device when no absorption is present(theoretical) (12) compared to the results from a microfluidic devicefabricated from an absorbing material—PDMS, and a low-absorbingmicrofluidic device (13) fabricated from COP. Both the COP microfluidicdevice (13) and the plate culture have depletion kinetics that arelog-linear as would be expected, but only in the non-absorbingmicrofluidic device are the values close to those predicted byliterature in vivo values. The results from the absorbing microfluidicdevice, fabricated out of PDMS, are not only off from those predictedfrom literature values, but the shape of the graph is not log-linear, aswould be expected if metabolism was the only driver for compound loss.Indeed, the non-log-linear depletion of diazepam is a clear indicationof another dynamic for compound loss, namely the material absorptionthat is known to occur. FIG. 30 shows the predicated clearance ofDiazepam in vivo, on a plate, measured in an absorbing microfluidicdevice (12) fabricated from PDMS, and a low-absorbing, gas-impermeablemicrofluidic device (13) fabricated from COP. In summary, the plateculture underpredicts clearance, the absorbing microfluidic deviceoverpredicts clearance, and the non-absorbing microfluidic device, heretermed the “New Liver-Chip” accurately predicts intrinsic clearance.

As such, microfluidic devices fabricated out of a strategic combinationof gas-permeable and gas-impermeable materials are advantageous comparedto previously fabricated microfluidic devices as they decreaseabsorbency of important compounds being tested as well as allow theexperiments to access ambient gases.

The first component of the resulting invention, is a low-absorbenttwo-channel microfluidic device (1) comprising a gas-permeable membrane(7) between top (6) and bottom (8) channel layers, as well as agas-exchanger (9) to allow gas transport from the ambient environmentoutside the microfluidic device into the microfluidic device, in orderto meet the needs of the experiment. One embodiment of this invention isdepicted in FIG. 1, where a bonded, low-absorbent microfluidic device(1) may be seen. FIG. 2 depicts one embodiment of a possibleconfiguration of the layers, with the gas exchanger (9) on the bottom ofthe device, bonded to the bottom channel layer (8), bonded to themembrane (7), bonded to the top channel layer (6), bonded to the gasket(5). The organization seen in FIG. 2 is just one possible configuration.Any organization of the layers is considered, as long as the bottom andtop layers are separated by the membrane. The top (6) and bottom (8)channel layers, the membrane (7) and the gas exchanger (9) may beattached permanently or temporarily. In one embodiment the layers areattached through plasma-activated bonding. In one embodiment themicrofluidic device (1) is bonded permanently by coating themicrofluidic device (1) components with silane. In one embodiment, themicrofluidic device (1) is used for the characterization of organmicrobiomes. FIG. 3 depicts a cross-sectional view of one embodiment ofa low-absorbent microfluidic device contemplated herein. In FIG. 3depicts how in this embodiment the ports (2) in the gasket (5) line upwith the ports in the top channel layer (6). As well, FIG. 3 depicts howin this embodiment the top channel (3) in the top channel layer (6) isdirectly on top of the bottom channel (4) in the bottom channel layer(8), separated by the membrane (7). FIG. 3 also depicts an embodiment inwhich the membrane has membrane pores (10) and the gas exchanger has gasexchanger pores (11).

Microfluidic devices may be used to test the effects drugs, foods,chemicals, cosmetics, physiological stimulants stresses etc. have oncellular systems. In order to quantify metabolism of compounds in cells,such as liver cells, several factors should be understood, such ascompound interaction with biology, loss to materials, gradients acrossthe device, protein binding, update/efflux of transporters, passivediffusion through the membrane (7), as well as other possibleparameters. FIG. 9 depicts the drug development triangle.

FIG. 9 depicts the drug development triangle, comprising importantaspects of developing an understanding of how a therapeutic is going tointeract with the body. In summary, the study of pharmacokinetics aimsto understand how and quantitatively predict how a particular dose ormass of compound is processed by the various organs in the body toproduce and exposure concentration. Quantitative pharmacokineticsfocuses on the movement of pharmaceuticals in vivo and in vitro, such aspharmaceutical absorption, distribution, metabolism and excretion.Pharmacodynamics aims to understand and predict how that exposureconcentration results in a given effect (either efficacy or toxicity).Quantitative pharmacodynamics focuses on the effects of pharmaceuticalsin vivo and in vitro and the mechanism of their action. Examples ofpharmacodynamic studies include parent compound dose-response andmetabolite dose-response, focusing then on toxicity and efficacy of thepharmaceutical. Microfluidic devices can, have, and are being used tostudy both pharmacokinetics and pharmacodynamics, which underscores theimportance of understanding and controlling the concentration ofcompounds in microfluidic devices since concentration it is vital forboth fields, and, therefore, vital for understanding and predicting howa pharmaceutical is going to interact with the human body. Indeed, thebase of the drug development triangle, or the most basic data thatshould be collected during experimentation, is concentration—both interms of the effects the cells had on the concentration(pharmacokinetics) and the concentration the cells were exposed to(pharmacodynamics). To reiterate, to enable therapeutic prediction usingmicrofluidic devices and in vitro systems in general, there needs to bea high confidence in the concentrations of compound in the system and anunderstanding of how and why that concentration is changing.

Typical microfluidic devices for the use of studying cells are oftenfabricated out of entirely gas-permeable materials. These entirelygas-permeable microfluidic devices have the possibility of causingserious result variability, as gas-permeable materials tend to absorbsmall molecule compounds disrupting data. FIG. 11 depicts thesignature-compound distribution profile in an absorbing, gas-permeablemicrofluidic device (12), fabricated out of PDMS. The model depicts ahighly absorbing compound, midazolam, being perfused through both thetop and bottom channels of the microfluidic device 150 ul/hr. As seen inFIG. 11, only the cells closest to the port (2) where a compound, suchas a small-molecule pharmaceutical, is being introduced see the expectedconcentration or “dosed” concentration. The average concentrationimpinging on the cells differs from the input concentration, resultingfrom a concentration gradient along the length of the microfluidicdevice (12). As such, it is difficult to evaluate the compoundpharmacodynamics (e.g., EC₅₀, the concentration of drug that gives thehalf-maximal response from specimen) in the presence of flow andabsorption. To further complicate matters, the rate and level ofabsorption changes with exposure time. As such, spatio-temporalgradients will develop, which are extraordinarily difficult tocharacterize and account for. Indeed, it is as if a moving target(concentration changes along the length) is trying to be hit while thetarget is also changing in size (concentration changes with time).Absorption, especially, diminishes the ability of the system toaccurately predict toxicity and efficacy. FIG. 12 depicts astereotypical sigmoidal drug response curve and the influence ofabsorption on it. If it is assumed that the cells are in contact withall of the drug (or enzyme, etc.) entering the system, then it isassumed that the cells are metabolizing a resulting compound based onthe dose entering the system. However, if the cells are actually only incontact with lower levels of the drug (due to absorption or loss of thecompound to system components) then the effective concentration of drug(or enzyme, etc.) will be over-predicted. In other words, scientistswill believe that a higher than necessary amount of the drug (or enzyme,etc.) may be advantageous in order to produce a given effect. Theoveruse of the drug (or enzyme, etc.) due to microfluidic deviceabsorbency not only skews data and makes prediction unreliable, but alsoadds unnecessary costs for drug development and discovery. Moreimportantly, however, inaccurate predictions of EC₅₀ or TC₅₀, or theconcentration where 50% of a toxic effect is seen, could result in thepoor decisions on dosing concentrations for in vivo studies, includingclinical trials (based on the in vitro data). The basic principle oftoxicology is Sola dosis facit venenum or “the dose [is what] makes thepoison”. In other words, at a high enough concentration, most compoundswill become toxic; over-estimating the concentration that causestoxicity (TC₅₀) could result in erroneously dosing a patient with atoxic concentration of a compound. Safety assessment of absorbingcompounds, therefore, is seriously hampered by absorption.

FIG. 4 depicts an embodiment of a microfluidic device described in U.S.Pat. No. 8,647,861. The absorbent microfluidic device (12) wasfabricated with PDMS in one embodiment. PDMS, and similar fabricationmaterials, absorb highly many compounds that pharmaceutical scientistsdesire to test within the microfluidic devices.

Another important aspect of microfluidic device material choice istransparency. Transparency offers scientists the ability to imagemicrofluidic devices on microscopes and be able to get an intimateperspective on cellular interactions, phenotypes, and more. Opaquenessoffers scientists the ability to protect their experiments from ambientlight if necessary. As such, the microfluidic device may be partially orentirely transparent or entirely opaque depending on the requirements ofthe experiment.

The top channel layer (6) and bottom channel layer (8) comprisesubstrates containing channels, such as the top channel (3) and thebottom channel (4), or pathways for fluid movement and experimenthousing. Experiments contained within the channels include cell growthand testing. Channels, such as the top channel (3) and the bottomchannel (4), in the channel layers may be a variety of differentheights, including but not limited to equaling the height of the channellayer itself or cutting through the entire channel layer. At each end ofthe top channel is a port (2) or via so that fluids may be introducedinto the microfluidic device. As well, microfluidic deviceinfrastructure may be made to be in fluidic communication with themicrofluidic device through these ports (2). The top channel layer (6)and bottom channel layer (8) may be fabricated from the same ordifferent materials. In some embodiments these materials aregas-impermeable in order to limit compound absorbency. Gas-impermeablematerials that have also been shown to be low absorbing include cyclicolefin copolymer (CCP), cyclic olefin polymer (COP), polycarbonate,polyethylene (PE), polyethylene Terephthalate, polystyrene (PS), (PET)glass, etc. The top channel layer (6) and bottom channel layer (8) mayalso achieve gas-impermeability, and by default low absorption, by beingfabricated from a partially gas-impermeable material, coated with agas-impermeable substance, having its surface modified to reachimpermeability, etc.

The membrane (7) provides a diffusive barrier between the top channel(3) and bottom channel (4). While the membrane (7) may begas-impermeable, oftentimes it is beneficial to allow oxygen diffusionthrough the membrane (7). As such, in some embodiments, it is beneficialto have a gas-permeable membrane (7). For example, cell types in the topchannel (3) and bottom channel (4) may benefit from exchanging gases.Gas-permeability may be prioritized over low-absorbency in the membranelayer (7) for this diffusivity reason. In some embodiments, the membrane(7) may be a smaller volume as compared to the volumes of othercomponents of the microfluidic device (1), such as the top channel layer(6) and bottom channel layer (8) and the gas exchanger (9). If themembrane (7) has a smaller volume than other components it would notabsorb as much of the experimental compound, minimizing absorbencyimpacts. In other embodiments the membrane (7) is non-porous in order tolimit physical contact between top channel (3) and bottom channel (4)environments and inhabitants. In some embodiments, the membrane (7) maybe considered porous, containing membrane pores (10), in order to allowcontact between top channel (3) and bottom channel (4) environments andinhabitants. In one embodiment the membrane layer (7) is homogenous,such as being evenly porous across the entire layer. In anotherembodiment the membrane layer (7) is heterogenous, such as being porousonly in the regions that overlap top channel (3) and bottom channel (4)on the top channel layer (6) and bottom channel layer (8). In someembodiments the membrane (7) is flexible as to allow it to stretch. Inthis embodiment the ability to stretch is beneficial for experimentsinvolving cells attached to the membrane (7), as it is able to replicatemechanical strain on cells as seen in vivo. In some embodiments thisstretch is achieved by using vacuum in optional working channels (15),in the microfluidic device, such as those seen in FIG. 4 of theabsorbent microfluidic device. In one embodiment the working channels(32) have their own entrance ports (14). Using working channels (32) toinduce mechanical actuation and stretch of the membrane (7) creates astrain differential across the membrane (7) where strain in the centerof the microfluidic device (12) is significantly greater than the strainnear the ports (2). FIGS. 35A and 35B illustrate the difference instretch between the center of the membrane (7) and a section of themembrane (7) close to the ports (2) in a flexible absorbing microfluidicdevice that is stretched via vacuum application to the working channels.FIG. 35A demonstrates deformation of the channel due to engagement witha perfusion manifold assembly, even before stretching the membrane. FIG.35B shows this same device under stretch. It can be seen that in anabsorbing microfluidic device that is actuated in this manner, thatthere has is a non-uniform stretch profile along the channel length,especially but not limited to, the area toward the edges of the workingchannels and far away from the working channels.

FIG. 36 depicts the difference in stretch over the length of theabsorbing microfluidic device. In this embodiment of stretch, onlyapproximately 20% of the culture area is under the applied stretch basedon a preliminary study.

In some embodiments stretch is achieved by having a pressuredifferential across the top channel (3) and bottom channel (4), as topush the membrane (7) in the direction of the lower pressure channel.FIGS. 37A and 37B display the membrane (7) before and after the pressuredifferential is applied, in this case the pressure is applied to thebottom channel, causing the channel to deflect into the top channel.When stretch is not desired the inlet ports (2) may be pressurized andthe outlet ports (2) may not be pressurized. When stretch is desired thebottom ports (2) may then be pressurized so that the pressure in thebottom channel (4) is greater than that of the top channel (3). FIG. 38shows a side view of a 50 μm thick PDMS membrane (7), having hadfluorescent beads embedded in it, imaged on a confocal microscope atdifferent pressure differentials. The membrane deflects into the upperchamber of the microfluidic device. The fluorescent membrane wasfabricated by spin coating a layer of PDMS with fluorescent beads. Itmay be seen in FIG. 38 that the greater the pressure differential thegreater the level of stretch of the membrane (7). Confocal imaging ofthe beads showed a scatter plot for various levels of applied pressure.A curve was fit to the plot and compared to theoretical values. Theresults may be seen in FIG. 39, which indicates that a pressure of 3 kPacorresponds to a strain of about 4% for a PDMS membrane thickness of 50μm. The experiment was repeated for a 20 μm thick PDMS membrane (7).FIG. 40 shows 20 μm thick PDMS membrane (7) actuation imaged on aconfocal microscope. FIG. 41 shows a scatter plot for various levels ofapplied pressure versus measured strain across a 20 μm thick PDMSmembrane, with an expected linear curve fit for the pressure regimetested. FIG. 41, indicates that 3 kPa of applied pressure corresponds toa strain of about 11% for a membrane with a thickness of 20 μm. FIG. 42depicts strain from applied transmembrane pressure differentials usingvarious mathematical models to predict percent strain vs appliedpressure and plots vs actual data of different stretch regimes based onthe dominating physics. The model and data agree well, indicating athorough understanding of mechanism and forces experienced at themembrane. FIG. 43 depicts strain from applied transmembrane pressuredifferential in the “mechanical advantage region”—which is the pressurerange where the pressure range that is most physiologically relevant(i.e. pressure seen in vivo)—a zoomed in version of FIG. 42. Thesegraphs, taken together, indicate diminishing returns in regards tostrain achieved in the membrane for an applied pressure; as pressureincreases linearly, the additional amount of stretch begins to diminish.In the low-pressure regime, even small pressure yields a large change instrain. The range of expected data extracted from models and theexperiment data fit well as depicted in both FIGS. 42 and 43. Thisembodiment of actuation is compatible with the culture module previouslymentioned.

Actuating the membrane (7) via pressure differentials have severaladvantages over mechanically actuating the membrane via vacuum in theworking channels (32). First, microfluidic devices not containingworking channels are easier to fabricate. This embodiment of actuationin microfluidic devices may also be advantageous as it may be morephysiology relevant than other methods, which apply no pressure to thecell layer. Indeed, this stretching mechanism better recapitulates thephysiologic mechanisms for mechanical stretching of cells and tissues,which include pressure differentials. For example, arteries tend toexpand as the heart beats and expels blood from within the ventriclesand into the artery lumen. This expansion (and resulting strain on thecells composing the vasculature walls) occurs because of the pressuregenerated by the beating heart, much like a balloon expands whenpressurized with air. The pressures needed to flex the membrane andcreate these in vivo relevant strains is, in one embodiment, a similarpressure as would be seen in the capillary beds of the lungs. Statedmore simply, in one embodiment both the pressures that the cell layersare exposed to and the stretch are tuned to be simultaneouslyphysiologically relevant. Additionally, the shape of this stretch betteremulates the shape of the expansion seen in blood vessels and thealveolar sacs, since in this embodiment the membrane is physicallydisplaced into a channel and assumes the shape of an arc as opposed to alinear displacement (i.e. the membrane move up and down as itstretches). FIG. 44 depicts the physiologically relevant pressuredifferentials experienced at the endothelial-epithelial barriers asblood flows from large arteries, down to small capillaries, and theninto the larger venous vessels returning blood back to the heart. Sincemany Organ-Chips seek to model or mimic this epithelial-endothelialinterface, capturing the pressure differential that is experienced invivo can be quite advantageous for further recapitulating the mechanicalmicroenvironment. According to various sources, arteriolar capillarypressure in the pulmonary vasculature is approximately 3.3 kPa, with theinterstitial pressure being close to −0.8 kPa. In a particularembodiment of an Organ-Chip where the alveolus is modeled, the topchannel represents the alveolar interstitial with the bottom channelrepresenting the lung capillary beds. At an applied pressure of 3 kPa tothe bottom channel, not only is the pressure differential seen in vivoaccurately applied, but the resulting stretch of the membrane (˜11%)also accurately recapitulates the type of mechanical strain that wouldbe experienced in the alveolus due to the expansion of the lungs duringrespiration.

There are several methods to increase gas transport into microfluidicdevices. These methods include increasing fluid/media flow rate into themicrofluidic device, increasing dissolved gas content of the mediaflowing through the microfluidic device, and delivering gases to theinterior of the microfluidic device through the microfluidic device bulkmaterial.

In one embodiment, increased gas transport into the microfluidic devicemay be achieved by using higher flow rates of media containing theimportant gases, such as oxygen, into the microfluidic device. In thisembodiment, as the flow rate of the media is increases, more media isintroduced into the microfluidic device in a set amount of time, andthus more of the desired gas is introduced into the microfluidic device.The use of high flow rates in microfluidic devices to increase gastransport is useful in gas-impermeable microfluidic devices (13), as gasmay not diffuse into the microfluidic device otherwise. However,increasing the flow rate of media into the microfluidic device may notbe physiologically relevant, as fluids in vivo flow at specific flowrates and velocities depending on the vessel. It is usually desired toexpose specimen, such as cells, to similar conditions in vitro as isfound in vivo. Increasing the flow rate of media into the microfluidicdevice may expose specimen, such as cells, to undue levels of shear, forexample. It is extraordinarily disadvantageous in a microfluidicOrgan-Chip to be constrained to a certain flowrate by oxygen transport,as this is just one of a whole host of conditions that are trying to berecapitulated and may be at least in part controlled by flow rate.

In another embodiment, the dissolved gas content of the media flowingthrough the microfluidic device may be increased prior to it enteringthe microfluidic device. In one embodiment, the dissolved gas content ofthe media may be increased prior to entering the microfluidic device bybubbling gas through the media. In another embodiment, the dissolved gascontent of the media may be increased prior to entering the microfluidicdevice by pressurizing the media under a blanket of the desired gas to apressure higher than atmospheric pressure or with a concentration of aspecific gas that is higher than it is normally found in the ambientatmospheric environment. However, increasing the dissolved gas contentof the media may not be physiologically relevant as fluids in vivocontain specific concentrations of gas. Indeed, it has been demonstratedin the literature that exposure to excess oxygen concentrations cancause significant damage to tissues, due to the formation of reactiveoxygen species. It is usually desired to expose specimen, such as cells,to similar conditions in vitro as is found in vivo. Both of the priorembodiments, flowing media at higher flow rates and increasing thedissolved gas content of media, also succumb to a significant shortfall.As the media flows through the microfluidic device, the specimen at thebeginning of the channels will experience higher levels of the desiredgas than specimen at the outlet, since specimen at the beginning of thedevice will consume at least some, if not all of the gas flowing throughthe device. The specimen at the beginning of the channel may then uptakehigh levels of said gas, leaving lower levels of the desired gas forspecimen further downstream in the channels.

In order to overcome low levels of important gases in microfluidicdevices, as well as avoid the use of high flow rates and gasconcentrations of media, a gas exchanger (9) may be built into themicrofluidic device in such a way as to not promote small moleculecompound absorbency while still allowing important gases, such asoxygen, to diffuse uniformly through the microfluidic device. In oneembodiment the gas exchanger (9) is attached to the bottom of themicrofluidic device (1), such as to form a floor to the bottom channellayer. In this embodiment the ceiling of the bottom channel (4) would bethe cell culture membrane (7) and the base of the bottom channel (4)would be the gas exchanger (9). In one embodiment the gas exchanger (9)is a two-layer combination of PDMS and polyethylene terephthalate (PET).PDMS is gas-permeable and absorbent. PET is gas-impermeable andnon-absorbent. In one embodiment the PET may be porous, such ascontaining gas exchanger pores (11). In one embodiment the porosity iscreated through track etching. In one embodiment the porosity of the PETis between 0.1% and 50%. In this embodiment, track-etched PET or PCserves as a transparent scaffold to give the gas exchanger (9)mechanical stability and low-absorbency, while the thin layer ofgas-permeable PDMS seals the PET pores.

In another embodiment, a track-etched scaffold, conversely known as agas exchanger membrane, fabricated from a rigid polymer may be“silk-screened” with an elastomeric polymer. A track-etched scaffold orgas exchanger membrane fabricated from a rigid polymer, such as PET, maybe coated with an elastomeric polymer, such as PDMS, such that theelastomeric polymer permeates or impregnates the pores of the track. Thetrack-etched scaffold or gas exchange membrane may then be “squeegeed”or wiped to remove the excess elastomeric polymer. The elastomericpolymer may then be cured into the pores, such as to create asubstantially rigid gas exchanger with gas-permeable pores. Theadvantage here is that the volume of elastomeric polymer is minimized,and therefore absorption is minimized. The gas exchanger would almost bea composite material of the rigid polymer. The rigid material wouldcomprise a scaffold for holding small volumes of the elastomericpolymer.

Furthermore, the gas exchanger may be coated with or have a film of aparticular material in order to enhance bonding. For example, a gasexchanger comprising a porous, gas-impermeable substrate may not onlyhave the pores filled with a gas-permeable material, but may also have alayer or coating or film of the gas-permeable material on top of it.

“Like dissolves like” is a common expression used by chemists toremember how some solvents interact with solutes. It refers to “polar”and “nonpolar” solvents and solutes. For example, water is polar and oilis non polar. Like does not dissolve like well, meaning that water willnot dissolve oil. For example, water is polar and salt (NaCl) is ionic(which is considered extremely polar). Like dissolves like, that meanspolar dissolves polar, so water dissolves salt. Much the same, “likebonds to like.” It has been found that materials bond more easily, suchas through chemical treatment, plasma treatment, etc. For example, PDMSbonds easily to PDMS as compared to other polymers. As such, in oneembodiment, the gas exchanger may have a coating, or film, or layer,which allows it to more easily bond to other structures. FIG. 110 showsmultiple embodiments of a gas exchanger, some of which show saidcoating. In the embodiments shown, a substrate comprises regions whichare filled by another material. The regions may be pores. The pores maybe entirely or partially filled. Further, the pores may be filled aswell as covered, such as with a coating. The pores may be coated orcovered on one or both sides.

The combination of PDMS and porous PET provides gas exchangingproperties while having minimal absorption. In this embodiment some ofthe small molecule compounds may absorb into the PDMS through the poresin the PET, however compared to the gas exchanger (9) being fabricatedfrom an entirely absorbent material, this absorbency may be considerednegligible in many cases. Further in this embodiment of the gasexchanger (9), the porous, track-etched PET and PDMS gas exchanger (9)would not only be able to increase gas transport compared to acompletely gas-impermeable microfluidic device (13), but also decouplesgas transport from fluid flow. In another embodiment TeflonAF2400 may beused as a gas exchanger (9) material. TeflonAF2400 is an exceptionalmaterial, as it is transparent, gas-permeable and low-absorbing tonon-absorbing. In one embodiment, the gas exchanger (9) may befabricated out of a gas-permeable and/or gas-impermeable material andthen coated with TeflonAF2400. In another embodiment polymethylpentene(PMP), commonly called TPX, a trademarked name of Mitsui Chemicals, maybe used. TPX is another exceptional material, as it is transparent,gas-permeable and low-absorbing. Polymethylpentene (PMP) has severalother advantageous properties, such as favorable optical properties, alow cost, injection moldable, and resistant to many solvents. Resistanceto solvents may be important if the microfluidic device is to be usedduring assays, as assays often use harsh solvents. A resistance tosolvents may allow the microfluidic device to be used in a greater rangeof assays. FIG. 34 depicts some different varieties of gas-exchangers(9), including Teflon AF2400, TPX, and porous PET.

The theoretical delivery of oxygen to a microfluidic device via mediaflow alone, calculated based on the carrying capacity of water foroxygen at a flow rate of 30 μL/h, is 5.8 nmol/h. The theoretical maximumhepatocyte uptake rate of oxygen, calculated via literature valuesscaled to a microfluidic device seeded with liver cells, is 88 nmol/h.There is a discrepancy between these two values of 83.2 nmol/h, meaningthat the fluid flow does not provide sufficient oxygen to supporthepatocyte maintenance, metabolism, or other functions. If thehepatocytes do not receive enough oxygen, they will undergo apoptosis ornecrosis—they will die.

The theoretical oxygen flow rate in an absorbing microfluidic device(12) fabricated from PDMS is 574 nmol/h and was measured to be 225nmol/h±9.43 nmol/h, which is more than sufficient to supply even thehighly oxygen consuming hepatocyte cell type with sufficient oxygen. Thetheoretical oxygen flow rate through the bulk material in alow-absorbing, gas-impermeable microfluidic device (13) fabricatedprimarily from COP is 0 nmol/h and was confirmed via measurement ofoxygen transport to be 0 nmol/h±0.63 nmol/h. The theoretical oxygen flowrate in a low-absorbing, gas-permeable microfluidic device (1)fabricated from a strategic combination of gas-impermeable andgas-permeable materials and comprising a gas exchanger made from 11.3%porous PET is 65.2 nmol/h and was measured to be 21.8 nmol/h±6.74nmol/h, which is well-above the oxygen uptake rate of hepatocytes. Thetheoretical oxygen flow rate in a low-absorbing, gas-permeablemicrofluidic device (1) fabricated from a strategic combination ofgas-impermeable and gas-permeable materials and comprising a gasexchanger made from 40% porous PET is 231 nmol/h. The measured oxygenflow rate in a low-absorbing, gas-permeable microfluidic device (1)fabricated from a strategic combination of gas-impermeable andgas-permeable materials and comprising a gas exchanger made fromTeflonAF2400 was 48 nmol/h±1.80 nmol/h. The theoretical oxygen flow ratein a low-absorbing, gas-permeable microfluidic device (1) fabricatedfrom a strategic combination of gas-impermeable and gas-permeablematerials and comprising a gas exchanger made from TPX is 241 nmol/h andwas measured to be 265 nmol/h±40.9 nmol/h. All these delivery rates arewell in excess of the required oxygen delivery rate, as defined by thecellular oxygen uptake rate. The implication of this is that, oxygendelivery through the bulk material will not only supply a sufficientamount of oxygen as required for cellular function, but also willmaintain an oxygen saturated environment that is consistent along thefull length of the device.

The gas exchanger (9) may be built into other portions of themicrofluidic device (1) in other embodiments. In one embodiment the gasexchanger (9) is configured around the outer walls of the microfluidicdevice (1). In another embodiment the gas exchanger (9) interfaces withthe top channel layer (6) instead of the bottom channel layer (8) asdescribed in an above embodiment. In yet another embodiment, there aremultiple gas exchangers (9) configured in various locations in themicrofluidic device (1). Gas exchangers (9) may be built such that theymay be switched from gas-permeable to gas-impermeable at the scientistsliking in order to make the microfluidic device (1) more customizable.

Indeed, in embodiments where a porous PET scaffold is utilized, theporosity of the scaffold in large part defines the oxygen delivery ratethrough the bulk material. Therefore, by choosing a specific porosity,the oxygen delivery rate can not only be turned on and off in a binaryfashion, but also “tuned” to a variety of delivery rates depending onthe specifics of the application. Similarly, the location of the PETmembrane in a particular embodiment, can be chosen to selectively tunedgas exposure in each channel with a certain level of independence. Forexample, for Zone 1 of the human liver is exposed to high levels ofoxygen in vivo. A user might be advised to select a PET membrane of highporosity in this case. Conversely, Zone 3 in the liver is known to bepoor in blood oxygen levels. Here, the advisement would be to select amembrane with very low porosity to throttle oxygen delivery to the lowlevels seen in vivo. Similarly, cancerous tumors tend to create lowoxygen environments and a low porosity PET membrane might be advisedadhered to the top of the top channel component and the bottom of thebottom channel component. Conversely, to imitate the hypoxic environmentseen in the intestine, and specifically the colon, a high oxygenconcentration might be desired in the bottom channel, which representsthe vasculature, whereas a low oxygen environment would be advantageousin the top channel, which represents the intestinal lumen. To achievethis, a moderate porosity PET membrane might be chosen to be adhered tothe bottom of the bottom channel to delivery oxygen to the vasculature,and a non-permeable membrane chosen for the top of the top channel, tominimize oxygen transport through the bulk material and create thedesired hypoxic environment.

In some embodiments the microfluidic device has a gasket layer (5) onthe top with four ports (2) to interact with the ports (2) exiting thetop channel (3). The gasket (5) may be used to ensure a tight fluidicconnection between the microfluidic device (1) and relatinginfrastructure. In one embodiment the gasket (5) is made out of acompressible material. In another embodiment the gasket (5) is made outof an adhesive material. The gasket (5) may be used to keep themicrofluidic device (1) the same size as it's absorbent predecessor (12)in order to fit into existing microfluidic device accessories, such as aperfusion manifold. The gasket (5) may be embodied in multiple heightsin order to raise the height of the microfluidic device (1) to a desiredlevel such that it fits into a compression fit snugly. The gasket (5)may also be gas-impermeable so that it does not absorb any smallmolecule compounds into the walls of its ports (2). The gasket (5) mayachieve gas-impermeability and therefore, low absorbance, by beingfabricated from a partially or entirely gas-impermeable material, coatedwith a gas-impermeable substance, having its surface modified to reachimpermeability and low absorbance (such as plasma treatment), etc. Inone embodiment the gasket (5) covers the entire surface of themicrofluidic device (1). In another embodiment the gasket (5) onlycovers a portion of the surface of the microfluidic device (1).

In one embodiment the low-absorbing, gas-permeable microfluidic device(1) featuring a gas exchanger (9) may be used to introduce and sustain agas gradient in the microfluidic device (1). In this embodiment aspecific concentration of gas could be introduced to the gas exchanger(9). The gas is then depleted by the cell layers (33), such asendothelial and epithelial cell layers, resulting in a hypoxic topchannel (3) or luminal channel—or a gradient in gas from the bottom tothe top of the microfluidic device, which is consistent along the entirelength of the microfluidic device. In one exemplary embodiment the gasis oxygen. In another embodiment the gas is carbon dioxide. In anotherembodiment the gas is nitrogen. The gas gradient may be altered byintroducing cell layers (33) of various permeability. The verticalgradient of gas through the microfluidic device (1) maintains thelongitudinal concentration of gas along the entire length of themicrofluidic device (1). In the embodiment where an oxygen gradient isintroduced in the low-absorbing, gas-permeable microfluidic device (1)with a gas exchanger (9), the longitudinal oxygen concentration alongthe entire length of the microfluidic device (1) is maintained. FIG. 52depicts the method of introducing an oxygen gradient into thelow-absorbing, gas-permeable microfluidic device (1) comprising a gasexchanger (9), using said gas exchanger (9) to selectively introduce agas into the microfluidic device (1) from the vascular channel only,while creating a diffusive barrier to the oxygen-rich ambientenvironment. The channel comprising the organ specific cells may thenhave a lower, even anaerobic environment, such that bacteria (36), suchas Clostridium symbiosum, may thrive. In one embodiment, a gas-gradientis introduced into the low-absorbing, gas-permeable microfluidic device(1) by flowing the selected gas through adjacent working channels (32).In one embodiment, a gas gradient is introduced into the low-absorbing,gas-permeable microfluidic device (1) with a gas-exchanger (9) usingchemical reactions.

The advantage of the gas exchanger, as depicted in FIG. 52, is that thegas concentration within a microfluidic device may be done in a normalcell culture incubator, without the need for a specialized gas-controlincubator. While gas-control incubators may be used to control the gasconcentration of gas-permeable microfluidic devices, as shown in FIGS.117-125, many more laboratories solely have access to normal cellculture incubators, without gas-control. Therefore, the gas exchangerherein presented is highly enabling for those culturing cells that needgas environments other than atmospheric.

In one embodiment sensors may be used to measure the gas gradient in thelow-absorbing, gas-permeable microfluidic device (1). In the exemplaryoxygen gradient embodiment, oxygen sensors may be used to measure theoxygen gradient in the low-absorbing, gas-permeable microfluidic device(1). In one embodiment, the sensors are electrical sensors. In oneembodiment the sensors are optical sensors. In one embodiment, thesensors comprise a gas sensitive dye. In one embodiment, the gassensitive dye is an oxygen sensitive dye. In one embodiment the sensorsare external to the microfluidic device (1). In one embodiment, thesensors are embedded in the microfluidic device (1). In one embodiment,the sensors are in the top channel (3). In one embodiment, the sensorsare in the bottom channel (4). In one embodiment, the sensors are inboth the top channel (3) and the bottom channel (4).

Another embodiment of the present invention is an upgraded perfusionmanifold assembly (14) that minimizes the amount of small moleculecompound to absorb into its materials. The perfusion manifold assembly(14) may be seen in FIG. 7. In one embodiment, the perfusion manifoldassembly (14) comprises i) a cover or lid assembly (25) configured toserve as the top of ii) one or more fluid reservoirs (19), iii) agasketing layer (20) under said fluid reservoir(s) (19), iv) a fluidicbackplane (22) under, and in fluidic communication with, said fluidreservoirs (19), v) a capping layer (21) over said fluidic backplane(22), and vi) a projecting member or skirt (23) for engaging themicrofluidic device (1) or a carrier containing a microfluidic device(1).

Another embodiment of the present invention is an upgraded perfusionmanifold assembly that minimizes the amount of small molecule compoundto absorb into its materials. In one embodiment, the perfusion manifoldassembly comprises i) a cover or lid configured to serve as the top ofii) one or more fluid reservoirs, iii) a gasketing layer under saidfluid reservoir(s), iv) a fluidic backplane under, and in fluidiccommunication with, said fluid reservoirs, v) a capping layer over saidfluidic backplane, and vi) a projecting member or skirt for engaging themicrofluidic device or a carrier containing a microfluidic device.

The cover or lid assembly (25) may aid in protecting the reservoirs fromboth spilling and contamination. In one embodiment, the lid assembly(25) comprises a lid (15), filter(s), and a lid gasket (18). Filters maybe configured into the lid assembly (25) in order to aid in sterility ofthe fluid within the reservoirs (19). In one embodiment the filters areflat filters (16). These thin filters (16) may be cut from a flatsubstrate material. In one embodiment the filters are thick filters(17). These thick filters (17) may be cut from a thick substratematerial. In the embodiment wherein, the lid assembly (25) comprises alid gasket (18), the lid gasket may take on a variety of embodiments. Inone embodiment, the lid gasket is compressible. In one embodiment, thelid gasket is adhesive. The lid gasket may vary in thickness in order tobest seal the reservoirs (19) off from the external environment.Alternatively, in other embodiment, the lid gasket (18) comprises thefilters, instead of having separate filters. In one embodiment, the lidgasket (18) is porous. In another embodiment the lid gasket (18) isnon-porous. In one embodiment, the lid gasket (18) permanently conformsto the shape of the reservoirs (19) after the first time the reservoirs(19) is pressed into it. In another embodiment the lid gasket (18)temporarily conforms to the shape of the reservoirs after each time thelid gasket (18) is pressed onto them. In yet another embodiment, the lidgasket (18) does not conform to the shape of the reservoirs (19). Thecover or lid assembly (25) can be removed and the perfusion manifoldassembly (14) can still be used. In one embodiment, the lid assembly(25) is held onto the reservoir using a radial seal. An applied pressureis not necessarily required to create a seal. In another embodiment, thelid assembly (25) is held onto the reservoir using one or more clips,screws or other retention mechanisms.

The fluid backplane (22) may be used to route fluid from the reservoirsto the microfluidic devices, such as a microfluidic device. In oneembodiment, the perfusion manifold assembly (14) further comprisesperfusion manifold assembly ports (28) positioned at the bottom of thefluidic backplane. In one embodiment the fluidic backplane (22)comprises one or more fluidic resistors (27). In one embodiment, the oneor more fluidic resistors (27) are comprised of elongated, serpentinechannels. Without being bound by theory of any particular mechanism, itis believed that these resistors (27) serve to stabilize the flow offluid coming from the reservoirs (19) so that a stable flow can bedelivered to the microfluidic device (1), and/or they serve to provide ameans for translating reservoir (19) pressure to perfusion flow rate.

In previous renditions of this invention there has been a single cappingand gasketing layer (26) responsible for both capping and gasketing. Aprevious rendition may be seen in FIG. 8, which the invention presentedherein improves on. The invention presented here suggests two separatelayers. One for gasketing (20) and one for capping (21) the fluidicbackplane. In one embodiment both the fluid reservoirs (19) and fluidbackplane (22) are fabricated from hard plastics, and as such may need acompressible gasket (20) between them to protect from leaks at the sitesof fluid connections. Having two separate layers is advantageous assealing and compression may be decoupled—sealing does not requirecompression and likely does not require absorptive materials.Conversely, oftentimes materials having the characteristics necessary tobe used as gaskets, especially transparent gaskets, have absorbencyissues. In one embodiment both the capping (21) and gasketing (20)layers are transparent. It may advantageous to have transparent capping(21) and gasketing (20) layers so that the fluidic backplane (22) may beimaged on a microscope if necessary. In one embodiment of the newinvention, the gasketing layer (20) is made up of a compressiblematerial, such as SEBS, while the capping layer (21) is made up of anincompressible material, such as COP. In another embodiment, thegasketing layer (20) made up of a compressible material may be coated,such as with Parylene, in order to make it gas-impermeable. The cappinglayer may be partially or completely coated in Parylene. In an exemplaryembodiment, a partially coated capping layer fabricated out of COP isused in conjunction with a gasketing layer fabricated out of SEBS. Thecombination of a partially Parylene-coated COP capping layer and SEBSgasketing layer is advantageous over a single, completely Parylenecoated COP layer. Parylene is difficult to bond, whereas COP bonds wellto other materials, including other parts made out of COP. By using twolayers, one may seal the fluidic backplane to the Parylene-coated COPcapping layer by material bonding, and seal the capping layer to thereservoirs with the SEBS gasketing layer. Further, when using two layersonly a small piece of SEBS needs to be coated with Parylene tosuccessfully prevent absorption. If a single layer is used, anyfluid-contacting surface may need to be coated with Parylene, whichmeans that the ports, the face of the components being sealed (such asthe reservoirs), and the entire length of the fluidic routing channelsin the perfusion manifold assembly would need to be coated. Coating thatmuch of the COP capping layer is difficult. When Parylene is coated, thepart needs to be held somewhere, much like Achilles's heel. FIG. 69depicts a low-absorbing, gas-permeable microfluidic device where thechannel components are fabricated out of COP (which is known not toabsorb), the gasketing material is fabricated from PDMS with a Parylenecoating (which the coating is known not to absorb). In anotherembodiment, a perfusion manifold assembly microfluidic device carrierfor the use of interfacing the microfluidic device with a perfusionmanifold assembly is preferred. This embodiment of the microfluidicdevice is compatible with the face-sealing gasketing method in onepreferred embodiment of the device/perfusion manifold assembly.

In one embodiment the perfusion manifold assembly (14) comprises aprojecting member or skirt (23). In one embodiment, the projectingmember or skirt (23) is engaged with a microfluidic device (1). In oneembodiment, the microfluidic device (1) comprises a top channel (3), abottom channel (4), and a membrane (7) separating at least a portion ofsaid top channel (3) and bottom channel (4). In one embodiment, themicrofluidic device (1) comprises cells on the membrane (7) and/or in oron the channels. The projecting member or skirt (23) may be designed sothat the fluidic backplane (22) is able to easily align with aconnecting microfluidic device (1). In one embodiment, the projectingmember or skirt (23) may be designed in order to interact with a culturesystem.

The perfusion manifold assembly (14) may be attached together viaseveral methods. In one embodiment, screws (24) may be used to securethe perfusion manifold assembly (14). In another embodiment, clips areused to secure the perfusion manifold assembly (14). In anotherembodiment, adhesives are used to secure the perfusion manifold assembly(14). In another embodiment, surface modification is used to secure theperfusion manifold assembly (14). In one embodiment, the perfusionmanifold assembly (14) is permanently bonded together. In oneembodiment, the perfusion manifold assembly (14) is temporarily bondedtogether.

Experimental 1. Absorbency Experiments on Materials

A method for ascertaining the absorption of a specific small-moleculeinto a polymer was developed. The output of this method are thefundamental parameters that fully define the absorption of a specificcompound into the material tested; specifically, the diffusivity andpartition coefficient are ascertained. The test set-up is depicted inFIGS. 13A and 13B. The steps for ascertaining this absorption is asfollows:

-   -   1. Dissolve the small molecule in an aqueous phase (medium) and        incubate the solution (30) with the tested material (31), such        as in a vial (29). The incubation should be long enough so that        diffusion is not limiting absorption and the transport into the        material is at equilibrium with the transport of the compound        out of the material and into the aqueous phase.    -   2. Sample media from the vials at a number of time points.    -   3. Measure the concentration of the small molecule remaining in        the aqueous phase (30) using a mass spectrometer, plate reader,        etc. from the media sampled.    -   4. Curve-fit the measured data to quantify the absorption and        diffusion parameters.

Each experiment includes a number of controls and test conditions. Usingmultiple controls and test conditions allows absorption to the vial andwell-plate to be characterized, as well as absorption versus adsorptionto the tested material, as well as yielding the time-dependent nature ofabsorption into the material. Controls comprise vials (29) filled withsolely the small molecule dissolved in an aqueous phase (30) in order toquantify the loss of compound caused by adsorption to the glass of thevial (29). The goals of the experiments are to directly quantifypartitioning of the compound, or compound loss at equilibrium(kinetics), and to directly quantify diffusion of the compound, ortime-dependent compound loss (dynamics). The developed method is robustin regards to quantifying the drug-specific progression and extent ofcompound loss.

Single time point experiments are only capable of extracting kinetics,not dynamics. Time-dependent studies capture not only equilibriumendpoints (K), but also time-dependent changes/dynamics (D).One-dimensional computational models are used to fit experimentalresults of time-dependent studies. FIG. 14 depicts a finite elementanalysis model, or a computational model that is solved incrementally,of recovered compound concentration from a set volume of PDMS afterdifferent time points for compounds of varying diffusivity. The higherthe diffusivity the faster the compound absorbs into the surroundingpermeable material. The results show that the higher the diffusivity ofthe compound the lower the recovered concentration of the compound afterany time spent with a permeable material, such as PDMS. The results alsodemonstrate that the longer the time spent with a permeable material,such as PDMS, the lower the recovered concentration of the compound.Using a graph, like the one pictured in FIG. 14, experimental data canbe matched to one of the curves. Once the particular curve is known, theparameters which defined that curve are taken as the fundamentalparameters defining the material-compound absorptive interaction.

FIGS. 20A and 20B depict the results of absorption testing on manydifferent small molecule compounds such as pharmaceuticals, specificallyfor the parameter partition coefficient in PDMS. Many compounds weretested from multiple industry collaborators. Results in FIG. 76 depictthe level of absorption into PDMS and the material of the perfusionmanifold assembly (pod). Tested compounds include both approvedcompounds already on the market, as well as candidates still in thepharmaceutical development pipeline. The compounds cover a range ofmolecular weights and lipophilicity (log P), which are twophysicochemical parameters that indicate absorption. The results of thematerial testing were then plotted versus these parameters. The resultsshowed that the majority of small-molecules are at risk for significantPDMS absorption. However, the extent of absorption is not well-predictedby log P or molecular weight mathematical models alone, only stronglyindicative. It was found that approximately 60% of the compounds testedabsorb into PDMS, while none of the compounds absorb into COP.Surprisingly, it was found that approximately 50% of the compounds alsoabsorb into SEBS to some extent, a preferred material in one embodimentof the previously presented perfusion manifold assembly (14). Largemolecules, above about 1 kDa, have a low risk of absorption.

Midazolam is a small-molecule medication used for anesthesia, sedation,as a treatment for epilepsy, and as a sleep aid. Midazolam has a log Pvalue of 3.89, a PDMS partition value (K) of 201 and a SEBS partitionvalue (K) of 4.05. FIG. 70 shows the recovered concentration ofMidazolam from a solution that had been in contact with variousmaterials, including glass, polypropylene, polystyrene, PDMS, SEBS andCOP. The recovered concentrations were compared to the default dosingconcentration. Midazolam did not absorb significantly into glass,polypropylene, polystyrene or COP. Midazolam absorbed somewhat intoSEBS. Midazolam absorbed significantly into PDMS. FIG. 72 shows acomputational model of Midazolam absorbing into a high-absorbing,gas-permeable microfluidic device fabricated from PDMS. FIG. 72 showsthat only the cells at the beginning of the cell culture channel arecontacted by the drug before it is absorbed into the PDMS as the mediais perfused through the microfluidic device channel from left to rightin the image.

Bufuralol is a small-molecule beta blocker. Bufuralol has a log P valueof 3.5, a PDMS partition value (K) greater than 216, and a SEBSpartition value (K) of 4.77. FIG. 71 shows the recovered concentrationof Bufuralol from a solution that had been in contact with variousmaterials, including glass, polypropylene, polystyrene, PDMS, SEBS andCOP. The recovered concentrations were compared to the default dosingconcentration. Bufuralol did not absorb significantly into glass,polypropylene, polystyrene or COP as indicated by nearly 100% recoveryof the dosed compound. Bufuralol absorbed somewhat into SEBS. Bufuralolabsorbed almost entirely into PDMS—so much so that the recoveredconcentration from the PDMS experiments was below the lower limit ofdetection of LCMS. The inability to detect the compound on the LCMShighlights the severity of the challenge of working with small moleculesin devices comprised of PDMS.

Material experiments were carried out with the drug Diazepam on bothPDMS and COP. FIGS. 15A and 15B depicts the absorption of the drugDiazepam into both materials PDMS and COP over time, based on therecovered concentration of Diazepam remaining in the fluid contained inthe glass vials where the material is contained. This depicts compound“loss” to the material over time. FIG. 15A depicts the differencebetween dosing concentration and compound recovery from the solutioncontaining Diazepam when in contact with PDMS for up to 72 hours. Byhour 12 almost two thirds of the Diazepam had been absorbed by the PDMS.Computational modeling is also shown in FIG. 15A to match samples takenat seven time points. FIG. 15B depicts the difference between dosingconcentration and compound recovery from the solution containingDiazepam when in contact with COP for up to 72 hours. Over the course of72 hours there was minimal, if not no, absorption into COP. Theexperiments emphasize the large absorbance difference between PDMS andCOP.

Material coatings were also tested to gauge their effectiveness inprotecting commonly used microfluidic device construction materials fromabsorption. Parylene is a trade name for a variety of poly(p-xylylene)polymers that may be used to coat materials via chemical vapordeposition. Parylene is of interest, as Parylene coated materials, suchas PDMS or SEBS, may be effectively used to construct low-absorbing, yetflexible microfluidic devices since while the layer of depositedParylene is rigid, it is thin enough to allow the flexibility of thematerial underneath to remain flexible.

Parylene-coated PDMS gaskets were exposed to the fluorescent moleculeRhodamine B and fluorescently imaged. FIG. 31 depicts microscopy imagesof the Parylene-coated PDMS gaskets after having been exposed toRhodamine B. Only a slight pinkish hue is visible and only on some ofthe corners, indicating some absorption is present but localized toareas that might not have been fully coated. However, the absorption isprimarily localized to areas with sharp corners. No absorption was seenwithin the microfluidic device ports leading into the channels, theactual region that is required be low-absorbing. Initial qualitativeanalysis of Parylene coating was found to be promising.

Parylene-coated SEBS gaskets were exposed to the fluorescent moleculeRhodamine B and fluorescently imaged as well. FIG. 32 depicts microscopyimages of the Parylene-coated PDMS gaskets after having been exposed toRhodamine B. A slight pinkish hue is visible, indicating some minimalabsorption is present. However, the absorption is primarily localized toareas with sharp corners. Some absorption can be seen inside of the via,but it was minimal, difficult to visualize, and quite possibly anoptical artifact unrelated to absorption.

Two quantitative studies were run on Parylene coated materials in orderto assess its effectiveness in minimizing small molecule absorbency. Inthe first study Parylene coated SEBS and Parylene coated PDMS were bothexposed to Rhodamine B and Coumarin. In the second study the absorptionof Parylene coated SEBS and Parylene coated E140 were compared to theabsorption of known low-absorbing materials, such as glass and COP.

In the first round of absorption studies, SEBS and PDMS gaskets werecoated with Parylene at two thicknesses: 2 μm and 8 μm. Parylene-coatedgaskets were exposed to Rhodamine B and Coumarin for 0, 14, 40 and 72hours. The remaining concentration of Rhodamine B and Coumarin in theexposure solution were measured on a plate reader. Each condition wastested on two gaskets. Limited replicates were available due to thenumber of conditions run. This “shotgun approach” was used in order totry many coating conditions and quickly determine the best options. FIG.33A shows the results of studies on absorption into Parylene coatedmaterials and depicts the fraction of Coumarin recovered from thesolutions. FIG. 33B depicts the fraction of Rhodamine B recovered fromthe solutions for coatings of varying thickness on two materials knownto absorb. FIG. 33A shows that some Coumarin was absorbed by both thecoated PDMS and SEBS with different coating thicknesses. FIG. 33B showsthat minimal Rhodamine B was absorbed by the PDMS and SEBS at thedifferent coating thicknesses. One observation from the experiment wasthat the Parylene may crack, leading to gasket absorption. Anotherobservation from the experiment was that Parylene adhesion to the PDMSand SEBS was poor, resulting in easy removal of Parylene. Finally, partswere difficult to handle, as the Parlene coating was extremelyhydrophobic, and thus “slippery.” These coating issues may be absolvedby optimizing the masking strategy to prevent cracking or tearing priorto mask removal, optimizing the geometry of the gaskets to remove sharpedges and reducing the bulk gasket volume so that just the functionalcomponents of the microfluidic device interfaces with the perfusionmanifold assembly instead of the gasket. Regardless, Parylene was shownto improve the absorbency issues of both PDMS and SEBS.

In the second round of absorption studies, after parylene-coatingprocess optimization, the absorption of Parylene coated SEBS andParylene coated E140 were compared to both the absorption of knownlow-absorbing materials, such as glass and COP, and a control solutionof the drug not in contact with a material. The coated materials wereexposed to a solution carrying a known concentration of the drug,Coumarin. The solution was tested three times to quantify the remainingconcentration of the Coumarin, before exposure to the material, at 22hours and at 92 hours. The results of the experiment showed that theglass and COP did not absorb, when compared to the control solution. Theresults of the experiment showed that uncoated SEBS and E140 both absorbsmall molecules. SEBS absorbed more of the compound than E140. Theresults of the experiments show that materials coated with Parylene donot absorb significant amounts of small molecules. FIG. 68 shows theresults of the experiment and only non-coated materials were seen toabsorb in this experiment.

2. Absorbency Experiments on High-Absorbing, Gas-Permeable MicrofluidicDevices

A computational absorption model of a microfluidic device comprising atop channel (3), a bottom channel (4), and a membrane (7) separating atleast a portion of said top channel (3) and bottom channel (4) wasbuilt. The model allows different variables to be changed, includingpermeability of the material (D) and absorbance of the material orpartition coefficient (K) (which are both deduced from the materialtesting experiments), flow rate of the fluid, diffusivity of thecompound in the fluid, geometry of the microfluidic device channels andmaterial, cellular phenomena like active and passive transport as wellas metabolism, etc. A depiction of the computational model of amicrofluidic device comprising a top channel, a bottom channel, and amembrane separating at least a portion of said top channel (3) andbottom channel may be seen in FIG. 17. The absorption models may bevalidated with commonly used or tool compounds. Stand-alone absorptionexperiments proved predictive of drug absorption. The ability tomathematically model drug absorption is useful in designing experiments,including permeability of the material, absorbance of the material, flowrate of the fluid in the top and bottom channels, diffusivity of thecompound in the fluid, etc. Understanding an experiment, and the likelyresults, before the experiment is carried out enables scientists tobetter economize funds and time.

Absorption modeling to inform experimental design was tested using thecompound Coumarin. Coumarin was flowed through an absorbing microfluidicdevice (13) and the recovered concentration in the bottom channel wassampled. The experiment was run at two different flow rates, 60 μL/hr asseen in FIG. 18A and 150 μL/hr as seen in FIG. 18B. The results of theexperiment not only showed that less compound is absorbed into PDMS atfaster flow rates, but also that the absorption modeling correctlyhypothesized the results within a reasonable degree of error, validatingthe approach.

However, computational models are oftentimes not always enough.Computational models may not work at all, as some compounds absorbcompletely. Indeed, if models are used to correct data from an absorbingmicrofluidic device experiments (with cells), the models will not beable to account for total absorption. That is to say, if cells areexposed to a very low concentration of compound, even if we can predictthis exposure level, it may be too low to be a useful correction.Regardless of the ability to correct data in only some situations,computational models also may require a complicated workflow. In orderfor computational modeling to work, absorption of every compoundintroduced into the system should be quantified first in materialcharacterization studies. As well, running multiple computational modelsbefore every experiment to design the experiment to minimize absorptionand then running an additional set of models to correct or account forabsorption that did occur is not sustainable, especially for large scaleexperiments with many conditions. As well, computational models may notbe able to accurately deconvolute data in cell-based experiments due tohigh numbers of variables, including those introduced by the cells. Forexample, concentration gradients due to absorption along the length ofthe absorbing microfluidic device and the fact that the concentrationwill also be changing with time makes concentration a “moving target.”Even with the aid of computational models to account for many of thesevariables, in the presence of absorption there is still a decreasedoverall confidence in results in in vitro to in vivo extrapolation(IVIVE).

FIG. 19 depicts the complexity of modeling and understanding thedynamics of compound deposition in the interior of an absorbingmicrofluidic device (13) related just to cellular functions that changethe concentration of a compound within the device. Indeed, even withoutthe added complexity of absorption, the dynamics of such a microfluidicdevice are challenging to model because this may includebiological/physiological factors such as passive cellular permeability,metabolism, and transport across the membrane.

FIG. 29A depicts an expected depletion model of the drug Diazepam in aplate culture calculated from in vivo drug clearance data versus actualdata collected from a plate culture. FIG. 29B depicts an expecteddepletion model of the drug Diazepam in a microfluidic device when noabsorption is present (theoretical) (12) compared to the results from amicrofluidic device fabricated from an absorbing material—PDMS, and alow-absorbing microfluidic device (13) fabricated from COP. Both the COPmicrofluidic device (13) and the plate culture have depletion kineticsthat are log-linear as would be expected, but only in the non-absorbingmicrofluidic device are the values close to those predicted byliterature in vivo values. The results from the absorbing microfluidicdevice, fabricated out of PDMS, are not only off from those predictedfrom literature values, but the shape of the graph is not log-linear, aswould be expected if metabolism was the only driver for compound loss.Indeed, the non-log-linear depletion of diazepam is a clear indicationof another dynamic for compound loss, namely the material absorptionthat is known to occur. FIG. 30 shows the predicated clearance ofDiazepam in vivo, on a plate, in an absorbing microfluidic device (12)fabricated from PDMS, and a low-absorbing, gas-impermeable microfluidicdevice (13) fabricated from COP. In summary, the plate cultureunderpredicts clearance, the absorbing microfluidic device overpredictsclearance, and the non-absorbing microfluidic device, here termed the“New Liver-Chip” accurately predicts intrinsic clearance.

Experimental outputs included concentration, C (μM) and time, t(minutes). Rate of reaction, k_(e), was then calculated using theequation:

$k_{e} = \frac{\ln\left( \frac{C_{1}}{C_{2}} \right)}{\left( {t_{2} - t_{1}} \right)}$

Chip clearance (CL), a measure of the ability of the microfluidic deviceto remove compound from the media passing through, was then calculatedusing the equation:

CL _(Media)

Intrinsic clearance (CL_(int)), the ability of an organ to removecompound from the blood passing through it, was then calculated usingthe equation:

${CL}_{int} = {\frac{CL}{f_{u_{media}}}*\frac{\#\;{Cells}_{organ}}{\#\;{Cells}_{chip}}C}$

The governing equation for intrinsic clearance, which is consistent withmethods previously published in literature for determination ofclearance in an in vitro system, is then:

${CL}_{int} = {\frac{\ln\left( \frac{C_{1}}{C_{2}} \right)}{\left( {t_{2} - t_{1}} \right)}*V_{Media}*\frac{1}{f_{u_{media}}}*\frac{\#\;{Cells}_{organ}}{\#\;{Cells}_{chip}}}$

Microfluidic device clearance was quantified as a function of the parentcompound depletion. In vivo values were used for comparison to Diazepamhepatic intrinsic clearance or CL_(int) values obtained from the twomicrofluidic device types. PDMS microfluidic device values were found tobe artificially high due to absorption, which causes compound loss thatis erroneously attributed to metabolism. As such, there was anoverestimation of clearance in PDMS microfluidic devices. Plate culturevalues were significantly lower than in vivo values due to anunderprediction of clearance.

FIG. 72 depicts a computational experiment wherein a solution containingthe drug Midazolam was flowed through a high-absorbing, gas-permeablemicrofluidic device (12) fabricated from PDMS at 150 μL/hr for the shortduration of a few hours. It may be seen in FIG. 72 that only the cellsat the beginning of the channel see the Midazolam, as the PDMS rapidlyabsorbs the drug such that cells later in the channel are unable tointeract with the drug. Further, it is more biologically relevant, andmore cost effective to use lower media flow rates, such as 30 μL/hr. Atthese lower flow rates, even fewer cells would come into contact withthe small molecule drug, as the media would be in contact with absorbingmaterial at the beginning of the channel for longer periods of time,when compared to media at higher flow rates. Using microfluidic devicesfabricated from absorbing materials, such as PDMS, could lead to anoverestimation or underestimation of in vivo metabolism by as much as100-fold depending on what is quantified to determine metabolism. Ifdepletion of a compound is used to estimate metabolism, then metabolismwould be overestimated. If quantification of a metabolite is used as areadout of metabolism, then metabolism would be underestimated. Further,it is difficult to know how much metabolism is being over or underestimated, as compound-material interactions and flow rates also play apart in the understanding of the metabolizing system. For midazolamspecifically, for high flow rates, where metabolite quantification wasused as a readout, there has been a consistent under-estimation ofmetabolism by anywhere between 10-fold and 100-fold, with greaterunder-estimation for lower flow rates. It is suggested thatlow-absorbing microfluidic devices would accurately estimate in vivodrug metabolism, assuming a rate of metabolism in the microfluidicdevice is similar to that seen at in vivo.

FIG. 22 depicts the COMSOL computational model of the absorbingmicrofluidic device (12).

A two-dimensional computational model was created that represented anabsorbing microfluidic device (13), fabricated from PDMS and containingtwo cell layers (33). The microfluidic device comprised a top channel(3), a bottom channel (4), and a membrane (7) separating at least aportion of said top channel (3) and bottom channel (4). A representativesmall molecule compound was dosed only in the bottom channel (4).Absorption is minimized when the bottom channel (4) is dosed instead ofthe top channel (3), as the PDMS bulk on the bottom channel layer (8) isthinner than the PDMS bulk on the top channel layer (6). As there isless PDMS on the bottom channel layer (8), there is less volume forsmall molecule compounds to absorb into.

After computational models of the microfluidic devices discussed hereinwere created and analyzed, physical laboratory experiments wereconducted in order to assess absorption in to microfluidic devicescomprising cell layers.

A low-absorbing, gas-impermeable microfluidic device (13) fabricatedfrom COP, an absorbing microfluidic device (12) fabricated from PDMS anda cell culture plate were seeded with various liver cells, includinghepatocytes, in order to assess liver cell viability and function. FIG.28A depicts liver cells in a low-absorbing, gas-impermeable microfluidicdevice (13) fabricated from COP on day 7 of culture. FIG. 28B showscomparable albumin production in the liver cells in both thelow-absorbing, gas-impermeable microfluidic device (13) fabricated fromCOP and the high-absorbing, gas-permeable microfluidic device (12)fabricated from PDMS. Albumin production in the plate culture wassignificantly lower than in both of the microfluidic devices. Protocolsto increase oxygen delivery to hepatocytes were used in order to createthe data shown in FIG. 28B. One such protocol includes increasing theflowrate entering the microfluidic devices.

Experiments were also run to assess whether high flow rates in the topand/or bottom channels of the microfluidic device impact absorption intothe bulk material of the microfluidic device. Four conditions ofmicrofluidic devices were seeded with two types of human liver cells,Hepatocytes and LSEC, and delivered oxygen through higher flow rates inthe bottom or basal channel. Oxygen delivery to cells layers (33) inmicrofluidic devices is of great importance, as the cell layers (33)oftentimes demand a particular oxygen concentration or rate of deliveryin order to survive and/or function. In some embodiments, cell layers(33) may need high levels of oxygen. In other embodiments, cell layers(33) may need very low levels of oxygen. The microfluidic devices testedinclude: five low-absorbing, gas-impermeable microfluidic devices (13)fabricated from COP with top channel flow rates of 0 μL/hr and bottomchannel (4) flow rates of 300 μL/hr; five low-absorbing, gas-impermeablemicrofluidic devices (13) fabricated from COP with top channel (3) flowrates of 10 μL/hr and bottom channel (4) flow rates of 300 μL/hr; fiveabsorbing, gas-permeable microfluidic devices (12) fabricated from PDMSwith top channel (3) flow rates of 10 μL/hr and bottom channel (4) flowrates of 30 μL/hr; and five high-absorbing, gas-permeable microfluidicdevices (12) fabricated from PDMS with top channel (3) flow rates of 10μL/hr and bottom channel (4) flow rates of 300 μL/hr. All microfluidicdevices had human hepatocytes seeded in the top channel (3) and humanLSECs seeded in the bottom channel (4). All microfluidic devices wererun on syringe pumps as opposed to culture modules. One question to beanswered by the experiments was whether the microfluidic devicessupported liver cell viability and function. Experiment readoutsincluded phase imaging, albumin production, CYP540 production and RNAendpoint analysis.

FIGS. 55A, 55B and 55C show hepatocyte attachment and morphology in botha low-absorbing, gas-impermeable microfluidic device (13) fabricatedfrom COP and a high-absorbing, gas-permeable microfluidic device (12)fabricated from PDMS on day 1, day 2 and day 3 of cell layer (33)growth. FIG. 55A shows hepatocyte attachment and morphology in alow-absorbing, gas-impermeable microfluidic device (13) fabricated fromCOP on day 1. FIG. 55B shows hepatocyte attachment and morphology in alow-absorbing, gas-impermeable microfluidic device (13) fabricated fromCOP on day 2. FIG. 55C shows hepatocyte attachment and morphology in alow-absorbing, gas-impermeable microfluidic device (13) fabricated fromCOP on day 3. FIG. 56A shows hepatocyte attachment and morphology in ahigh-absorbing, gas-permeable microfluidic device (12) fabricated fromPDMS on day 1. FIG. 56B shows hepatocyte attachment and morphology in ahigh-absorbing, gas-permeable microfluidic device (12) fabricated fromPDMS on day 2. FIG. 56C shows hepatocyte attachment and morphology in ahigh-absorbing, gas-permeable microfluidic device (12) fabricated fromPDMS on day 3. On days 1, 2, and 3 hepatocyte attachment and morphologywere similar in both microfluidic device designs.

FIGS. 57A and 57B show hepatocyte and LSEC morphologies on day 9 in ahigh-absorbing, gas-permeable microfluidic device (12) fabricated fromPDMS. FIG. 57A shows hepatocyte morphology on day 9 in a high-absorbingmicrofluidic device (12) fabricated from PDMS. FIG. 57B shows LSECmorphology on day 9 in a high-absorbing microfluidic device (12)fabricated from PDMS. FIGS. 58A and 58B show hepatocyte and LSECmorphologies on day 9 in a low-absorbing, gas-impermeable microfluidicdevice (13) fabricated from COP. FIG. 58A shows hepatocyte morphology onday 9 in a low-absorbing, gas-impermeable microfluidic device (13)fabricated from COP. FIG. 58B shows LSEC morphology on day 9 in alow-absorbing, gas-impermeable microfluidic device (13) fabricated fromCOP. Both hepatocytes and LSECs showed comparable morphologies andmaintained monolayers in both the low-absorbing, gas-impermeablemicrofluidic device (13) and the high-absorbing, gas-permeablemicrofluidic device (12) on day 9.

The portion of the experiment demonstrates a low-absorbing,gas-impermeable microfluidic device (13) can maintain the human livercell morphology, while still offering low-absorbency. Low-absorbency isadvantageous as it does not negatively impact small molecule studies asdo high-absorbency microfluidic devices (12).

FIGS. 59A and 59B show bile canaliculi fluorescence staining via MRP2 atday 9 of cell layer (33) culture on two different microfluidic devices.FIG. 59A shows bile canaliculi fluorescence staining via MRP2 on ahigh-absorbing, gas-permeable microfluidic device (12) fabricated fromPDMS using a 20× microscope objective on day 9 of cell layer (33)culture. FIG. 59B shows bile canaliculi fluorescence staining via MRP2on a high-absorbing, gas-permeable microfluidic device (13) fabricatedfrom COP using a 20× microscope objective on day 9 of cell layer (33)culture. There was similar development of bile canaliculi in both themicrofluidic devices fabricated from PDMS and COP, although neither wasideal. Ideal cell layers (33) would show interconnected networks.

FIG. 60 depicts an overview of albumin production across the fourconditions. The microfluidic devices tested include: five low-absorbing,gas-impermeable microfluidic devices (13) fabricated from COP with topchannel flow rates of 0 μL/hr and bottom channel (4) flow rates of 300μL/hr; five low-absorbing, gas-impermeable microfluidic devices (13)fabricated from COP with top channel (3) flow rates of 10 μL/hr andbottom channel (4) flow rates of 300 μL/hr; five absorbing,gas-permeable microfluidic devices (12) fabricated from PDMS with topchannel (3) flow rates of 10 μL/hr and bottom channel (4) flow rates of30 μL/hr; and five high-absorbing, gas-permeable microfluidic devices(12) fabricated from PDMS with top channel (3) flow rates of 10 μL/hrand bottom channel (4) flow rates of 300 μL/hr. Albumin levelssignificantly decreased in low-absorbing, gas-impermeable microfluidicdevices (13) fabricated from COP. The microfluidic devices without flowsuffered from lack of oxygen and non-physiologically relevant pHs due tofailure to properly buffer media by exposing the media with sodiumbicarbonate with the gas CO₂.

FIG. 61 shows CYP1A2 enzyme levels on day 14 following lysing of themicrofluidic devices. The high-absorbing, gas-permeable microfluidicdevices (12) fabricated from PDMS showed higher levels of CYP1A2 thanthe low-absorbing, gas-impermeable microfluidic devices (13) fabricatedfrom COP. The low-absorbing, gas-impermeable microfluidic devices (13)fabricated from COP lack much of the metabolic function seen in thehigh-absorbing, gas-permeable microfluidic devices (12) fabricated fromPDMS.

FIG. 62 shows CYP3A4 levels at day 14 following lysing of themicrofluidic devices. The high-absorbing, gas-permeable microfluidicdevices (12) fabricated from PDMS showed higher levels of CYP3A4 thanthe low-absorbing, gas-impermeable microfluidic devices (13) fabricatedfrom COP. The low-absorbing, gas-impermeable microfluidic devices (13)fabricated from COP lack much of the metabolic function seen in thehigh-absorbing, gas-permeable microfluidic devices (12) fabricated fromPDMS.

FIG. 63 shows CYP2A6 levels at day 14 following lysing of themicrofluidic devices. The high-absorbing, gas-permeable microfluidicdevices (12) fabricated from PDMS showed higher levels of CYP2A6 thanthe low-absorbing, gas-impermeable microfluidic devices (13) fabricatedfrom COP. The low-absorbing, gas-impermeable microfluidic devices (13)fabricated from COP lack much of the metabolic function seen in thehigh-absorbing, gas-permeable microfluidic devices (12) fabricated fromPDMS.

Seventeen microfluidic devices of various conditions where seeded withhuman liver cells in order to assess the effect of higher flowrates inthe apical or top channel (3). The microfluidic devices comprised: threelow-absorbing, gas-impermeable microfluidic devices fabricated from COPwith media equilibrated with 100% oxygen (i.e. 100 kPa, no CO2equilibration, with a 150 μL/hr flow rate in the top channel and a 150μL/hr flow rate in the bottom channel being run on a culture module;three low-absorbing, gas-impermeable microfluidic devices fabricatedfrom COP, with 21% oxygen media equilibration and 5% carbon dioxide, a150 μL/hr flow rate in the top channel and a 150 μL/hr flow rate in thebottom channel being run on a culture module; three low-absorbing,gas-impermeable microfluidic devices fabricated from COP, with mediaequilibrated to 21% oxygen and 5% carbon dioxide, a 150 μL/hr flow ratein the top channel and a 150 μL/hr flow rate in the bottom channel, andadditionally having 15 mM HEPES in the media to pH buffer the media,being run on a culture module; low-absorbing, gas-impermeablemicrofluidic devices fabricated from COP, with media equilibrated to 21%oxygen and 5% carbon dioxide, at a 300 μL/hr flow rate in the topchannel and a 300 μL/hr flow rate in the bottom channel being run on asyringe pump; two high-absorbing, gas-permeable microfluidic devicesfabricated from COP, with media equilibrated to 21% oxygen and 5% carbondioxide, with a 300 μL/hr flow rate in the top channel and a 300 μL/hrflow rate in the bottom channel being run on a syringe pump; and twohigh-absorbing, gas-permeable microfluidic devices, fabricated from COP,with media equilibrated with 21% oxygen and 5% carbon dioxide, with a 30μL/hr flow rate in the top channel and a 30 μL/hr flow rate in thebottom channel being run on a culture module. FIG. 64 shows anexperimental matrix in which all the experimental conditions for anoptimization study aimed at sustaining viability and function of livercells in microfluidic devices may be seen.

In total seventeen microfluidic devices, three culture modules and onesyringe pump were used. Three medias were used: WEM(−) 2% FBS; WEM(−) 2%FBS with 15 mM HEPES; and CSC 2% FBS. HEPES was tested in order toevaluate its cytotoxicity. The goal of the experiment was to test cellfunctionality as a reflection of oxygen perfusion within themicrofluidic devices. Timepoint analysis included bright field imaging,albumin secretion analysis, LDH secretion analysis, and CYP450 analysis.FIG. 65 shows albumin production at each condition shown in FIG. 64. Thegraph shows that there was an improvement in the albumin production inthe low-absorbing, gas-impermeable microfluidic device (13) fabricatedfrom COP when there was a higher flow rate in both the top channel (3)and bottom channel (4) as compared to when the higher flow rate wassolely in the bottom channel (4). Albumin production was about the samein the low-absorbing, gas-impermeable microfluidic device (13)fabricated from COP with top channel (3) and bottom channel (4) flowrates of 150 μL/hr as in the high-absorbing, gas-permeable microfluidicdevice (12) fabricated from PDMS with top channel (3) and bottom channel(4) flow rates of 30 μL/hr.

Protein binding in an absorbent microfluidic device (12) fabricated fromPDMS seeded with liver cells was also quantified for differentconcentrations of fetal bovine serum (FBS). Not only do compounds absorbinto materials, but proteins within the media may also bind to thecompound causing effective compound loss, since the compound is carriedpast the cells and they are not exposed to the compound. Diazepam wasused as the small molecule compound in these experiments. FIG. 16depicts the results of the experiment. The higher the concentration ofthe FBS, the lower the compound availability to the cells due to proteinbinding. The experiment is important for establishing available fractionof compound concentration for absorption in absorption experiments, butalso the fraction of compound available to cells, even withoutabsorption causing additional “loss”. Rapid Equilibrium Dialysis (RED)Devices were used to characterize binding. For media with 1% FBS, thecompound availability of Diazepam was 67%. Protein binding data was usedto convert the rate of metabolism to intrinsic clearance as seen in theequations above.

Reciprocation of cell culture media was tested in order to assess thepotential benefits, including oxygenation of media and ensuring thatcells see the full dosing concentration of drug in a small volume ofmedia. FIGS. 66A, 66B and 66C show an experimental setup forreciprocation of media. The setup involves pumping media through alow-absorbing, gas-impermeable microfluidic device (12) fabricated fromCOP using a syringe pump (38). The media collects in an externalreservoir that is connected to the outlet port (2). Once most of themedia has been pumped out of the syringe (37), the syringe pump (38)reverses direction and begins to pump media from the external reservoir(39) back into the syringe (37). In the process of pumping the mediaback and forth, in one embodiment the media flows through gas-permeabletubing, which allows ambient gases to access the media. In anotherembodiment, the media that has collected in the outlet reservoir isexposed to the ambient atmospheric environment allowing it to rapidlyequilibrate to the gas concentrations in the air, in this case supplyingthe needed oxygen levels for the cells to function properly. Becausethis reservoir is “open” to the external environment, the media is ableto equilibrate to the ambient oxygen concentration in the air. If thecells in the device have depleted the oxygen in the media, oxygen willquickly diffuse into the media to re-saturate with dissolved oxygen. Theexperimental setup is not only low-absorbing, but also importantlydecreases system volume. FIG. 67 depicts the flow process of theexperimental setup shown in FIGS. 66A, 66B and 66C, where the media ispushed back and forth through the microfluidic device (13) from thesyringe (37) and external reservoir (39), which exposes the media to therequired gas concentrations. In FIG. 67, the media is first drawn fromthe external reservoir, through the microfluidic device, into thesyringe. The media is then optionally held static in the syringe in themiddle panel of the figure. The media is then pushed out of the syringe,back through the microfluidic device, into the external reservoir. Theexternal reservoir may alternatively be known as a reservoir or fluidreservoir.

3. Absorbency Experiments on Low-Absorbing, Gas-Permeable MicrofluidicDevices

Three of each of four different types of microfluidic devices wereseeded with different varieties of liver cells to form a “Liver-On-Chip”or “Liver Chip” in order to assess viability in different microfluidicenvironments. The top channel (3) was seeded with human hepatocyte cellsand the bottom channel (4) was seeded with human sinusoidal endothelialcells. The first condition was an absorbing microfluidic device (12)described in U.S. Pat. No. 8,647,861 fabricated from PDMS. Theabsorbing, PDMS microfluidic device (12) represented a negative control.The second condition was a low-absorbing, gas-impermeable microfluidicdevice (13) fabricated from COP. The gas-impermeable, low-absorbingmicrofluidic device (13) represented a positive control. The thirdcondition was a low-absorbing, gas-permeable microfluidic device (1)comprising an 11% porous PET scaffold and PDMS thin film gas exchanger(9). The fourth condition was a low-absorbing, gas-permeablemicrofluidic device (1) comprising a PDMS thick film gas exchanger (9)but no porous PET scaffold. Media was flowed through the microfluidicdevices at 30 μL/hr. Functional readouts of the experiment includedmorphology, albumin production, and bile canaliculi structure.Morphology was determined with brightfield imaging. Albumin productionwas quantified with effluent collection and ELISA tests. The presence ofa proper bile canaliculi structure was evaluated with immunofluorescenceto visualize MRP2 expression. Slits were cut in the tray of the culturemodule used in order to achieve better oxygen transport through the gasexchanger (9). FIG. 45A depicts a gas-permeable, low-absorbingmicrofluidic device (1) comprising an 11% porous PET and PDMS thin-filmgas exchanger (9). FIG. 45B depicts a low-absorbing, gas-permeablemicrofluidic device (1) comprising a PDMS thick film gas exchanger (9).

FIGS. 46A, 46B, 46C and 46D depict the morphology of the cell monolayer(33) in an absorbing microfluidic device (12). FIG. 46A shows themonolayer (33) on Day 1. FIG. 46B shows the monolayer (33) on Day 3.FIG. 46C shows the monolayer (33) on Day 6. FIG. 46D shows the monolayer(33) on Day 10. The monolayer (33) appeared to be maintained through Day10, with slight morphological decline.

FIGS. 47A, 47B, 47C, and 47D depict the morphology of the cell monolayer(33) in a low-absorbing, gas-impermeable microfluidic device (13)constructed from COP. FIG. 47A shows the monolayer (33) on Day 1. FIG.47B shows the monolayer (33) on Day 3. FIG. 47C shows the monolayer (33)on Day 6. FIG. 47 D shows the monolayer (33) on Day 10. The monolayer(33) appeared to be declining rapidly over the course of the 10 days,with most cells completely dead or dying by Day 10.

FIGS. 48A, 48B, 48C and 48D depict the morphology of the cell monolayer(33) in a low-absorbing, gas-permeable microfluidic device (1) with aporous PET and thin film PDMS gas exchanger (9). FIG. 48A shows themonolayer (33) on Day 1. FIG. 48B shows the monolayer (33) on Day 3.FIG. 48C shows the monolayer (33) on Day 6. FIG. 48D shows the monolayer(33) on Day 10. The monolayer (33) appeared to be maintained through Day10, with slight morphological decline (similar to the gas-permeable, butabsorbing device in FIG. 46A-D).

FIGS. 49A, 49B, 49C and 49D depict the morphology of the cell monolayer(33) in a low-absorbing, gas-permeable microfluidic device (1) with athin film PDMS gas exchanger (9). FIG. 49A shows the monolayer (33) onDay 1. FIG. 49B shows the monolayer (33) on Day 3. FIG. 49C shows themonolayer (33) on Day 6. FIG. 49D shows the monolayer (33) on Day 10.The monolayer (33) appeared to be maintained through Day 10, with slightmorphological decline (similar to the gas-permeable, but absorbingdevice in FIG. 46A-D).

FIGS. 50A, 50B, 50C and 50D depict the MRP2 signal of the BileCanaliculi of all the conditions at Day 14. FIG. 50A shows the BileCanaliculi MRP2 signal on an absorbing microfluidic device (12)constructed from PDMS on Day 14. FIG. 50B shows the Bile Canaliculi MRP2signal on a low-absorbing, gas-impermeable microfluidic device (13)constructed from COP on Day 14. FIG. 50C shows the Bile Canaliculi MRP2signal on a low-absorbing, gas-permeable microfluidic device (1) with aporous PET and thin film PDMS gas exchanger (9) on Day 14. FIG. 50Dshows the Bile Canaliculi MRP2 signal on a low-absorbing, gas-permeablemicrofluidic device (1) with a thin film PDMS gas exchanger (9) on Day14. There was no MRP2 signal for any of the conditions on Day 14.

FIGS. 51A and 51B depict average Albumin secretion in each of the fourconditions on Day 4, Day 9 and Day 13. Albumin secretion is lower inboth the low-absorbing, gas-permeable microfluidic device (1) with aporous PET and thin film PDMS gas exchanger (9) and the low-absorbing,gas-permeable microfluidic device (1) with a thin film PDMS gasexchanger (9) than the absorbing microfluidic device (12) constructedfrom PDMS. However, there is a significant improvement from thelow-absorbing, gas-impermeable microfluidic device (13) constructed fromCOP.

The absorbing microfluidic device (12) constructed from PDMS did notperform astonishingly well, however the cell layer (33) was alive at Day14. The low-absorbing, gas-impermeable microfluidic device (13)constructed from COP was surprisingly still alive at Day 1, however itunsurprisingly was dead at Day 14. Both the low-absorbing, gas-permeablemicrofluidic device (1) with a porous PET and thin film PDMS gasexchanger (9) and the low-absorbing, gas-permeable microfluidic device(1) with a thin film PDMS gas exchanger (9) showed improvement comparedto the low-absorbing, gas-impermeable microfluidic device (13)constructed from COP.

Experiments were also run to see if a low-absorbing, gas-permeablemicrofluidic device comprising a gas exchanger could be used to createoxygen gradients in the cell culture channels, also known as the top andbottom channels. A low-absorbing, gas-permeable microfluidic device (1)with a gas exchanger (9) was seeded with Caco-2 cells. The microfluidicdevice (1) was not seeded with endothelial cells. All media wasequilibrated in a 5% oxygen environment for 24 hours. A hypoxicincubator was set to maintain a 5% oxygen environment or 5 kPa partialpressure.

The proof-of-concept study demonstrates that the low-absorbing,gas-permeable microfluidic device (1) establishes oxygen micro-gradientsalong the height of the microfluidic device that support Caco-2epithelial grown and differentiation and a hypoxic environment in theapical chamber. FIG. 53 depicts Caco-2 morphology in the low-absorbing,gas-permeable microfluidic device, benefitting from the creation ofoxygen gradients from the vascular channel into the apical channel,which represents the intestinal lumen (1). FIG. 54 depicts the oxygenconcentration profile of the low-absorbing, gas-permeable microfluidicdevice (1) sampled at the four different ports (2): top channel (3)inlet port (2), top channel (3) outlet port (2), bottom channel (4)inlet port (2) and bottom channel (4) outlet port (2). Recreating themicro-anaerobic environments characteristic of the intestinal lumenenables first-in-kind co-cultures of mucosal host tissues with thepredominant fastidious commensal microbial species of the human gut. Anexample of fastidious commensal microbial species of the human gut isfirmicutes.

4. Absorbency Experiments on Perfusion Manifold Assemblies

Fluorescent molecule Rhodamine B (a fluorescent molecule that is alsomoderately absorbing into PDMS and SEBS) was dissolved in a buffer,flowed through a perfusion manifold assembly (14) and absorbingmicrofluidic device (12) fabricated out of PDMS at 30 μL/hr for 38 hourson a culture module. The perfusion manifold assemblies (14) were rinsedwith buffer not containing the fluorescent molecule at 200 μL/hr for anhour before the start of the experiment.

Following the experiment, the perfusion manifold assemblies (14) weredisassembled and the vias (35) of the capping, gasketing and backplaneassembly or fluidic layer assembly (34), as well as the perfusionmanifold assembly (14) resistors (27), were imaged with fluorescentmicroscopy. FIG. 25A depicts the resulting fluorescence in the fluidiclayer assembly (34) of an absorbing perfusion manifold assembly (14)comprising a combined gasketing and capping layer (26). FIG. 25B depictsthe resulting fluorescence on one aspect the invention described herein,a low-absorbing perfusion manifold assembly comprising both alow-absorbing capping and low-absorbing gasketing layer. In theembodiment of the perfusion manifold assembly (14) used in theexperiment, the capping layer was fabricated from COP and the gasketinglayer was fabricated out of SEBS coated with Parylene. Bright whitecolors in FIGS. 25A and 25B correlate to greater degree of absorption ofthe fluorescent molecule Rhodamine B.

FIG. 25A shows sample images of absorption of fluorescent moleculearound each of the four vias (35) in the fluidic layer assembly (34).FIG. 25B shows sample images of the little to no absorption of thefluorescent molecule around each of the four vias (35) in the fluidiclayer assembly (34).

FIG. 26 shows more comprehensive images of all of the experimentconditions. An absorbing perfusion manifold assembly (14) was tested. Asupposedly low-absorbing perfusion manifold assembly (14) was tested,comprising a COP capping layer (21) and a non-coated SEBS gasketinglayer (20) was tested. Five low-absorbing perfusion manifold assemblies(14), comprising a COP capping layer (21) and a Parylene coated SEBSgasketing layer (20) were also tested. FIG. 26 shows that the perfusionmanifold assembly (14) comprising a combined gasketing and capping layer(26) absorbed the fluorescent molecule. Bright white in the imagesindicate areas where the fluorescent molecule Rhodamine has beenabsorbed. FIG. 26 shows that the perfusion manifold assembly (14)comprising a COP capping layer and non-coated SEBS gasketing layerabsorbed the fluorescent molecule. The result is surprising, as it wasnot previously known that SEBS absorbed small molecules. FIG. 26 showsthat the perfusion manifold assemblies (14) comprising a COP cappinglayer and Parylene coated SEBS gasketing layer did not absorb asignificant amount of the fluorescent molecule.

FIGS. 27A and 27B show fluorescent molecule absorption in the resistors(27), having capped with SEBS and COP respectively. FIG. 27A shows thatthe resistors capped with SEBS surprisingly absorb fluorescentsmall-molecules to a relatively high extent. FIG. 27B shows that theresistors capped with COP absorb very little of the fluorescentsmall-molecule rhodamine. Note that in FIG. 27B the bright white linesrepresent an optical artifact (reflection of light by the walls of thechannel) as opposed to emission of Rhodamine fluorescence.

Perfusion manifold assemblies (14) comprising a low-absorbing cappinglayer (21) and low-absorbing gasketing layer (20) absorb significantlyless small-molecule than perfusion manifold assemblies (14) comprising asingle, absorbing capping and gasketing layer (26). This absorptionstudy demonstrates visually the importance of having perfusion manifoldassemblies fabricated from low-absorbing materials, such as COP, ortreated with low-absorbing coatings, such as Parylene.

Experiments were also run using the perfusion manifold assembly in itsentirety with microfluidic devices seeded with cell layers.

FIG. 10A depicts the absorption of a small molecule (Bupropion) invarious embodiments of the microfluidic system comprising of amicrofluidic device and perfusion manifold assembly, while FIG. 10Bdepicts the results of a test of that same compound in the same setupfor liver metabolism by the metabolizing enzyme CYP2B6. The apparentmetabolism of drug by liver cells in both an absorbent microfluidicdevice fabricated from PDMS and a gas-impermeable, low-absorbingmicrofluidic device fabricated from COP are depicted, demonstrating theeffects of absorption on the apparent rate of metabolism, whenquantified by production of a metabolite. It can be seen that the highlyabsorbing systems results in greater under-prediction of metabolism thanthe non-absorbing and lower-absorbing systems.

Oftentimes when cells come into contact with enzymes, they product asecondary compound which may then be used in the production of abiopharmaceutical. When the liver cells are able to access andmetabolize the enzyme CYP2B6 they produce the compound OH-Bupropion.Both the absorption of the enzyme into the microfluidic device andconnected infrastructure, as well as the formation of OH-Bupropion weremeasured. If the absorbency of microfluidic devices is ignored duringexperiments, then one would assume that cells were in contact with theconcentration of enzyme that was dosed into the microfluidic device.However, if the bulk material of the microfluidic device is absorbingthe enzyme, then it would appear as though the cells are under-producingexpected compounds when in contact with the enzyme.

The results speak to a significant under-prediction of OH-Bupropionmetabolism in the test-setup comprising an absorbent microfluidic device(12), the perfusion manifold assembly (14) comprising the combinedgasketing and capping layer (26), and the culture module. When thevariability of enzyme absorption into the bulk of the microfluidicdevice is eliminated from the experiment, such as using a low-absorbing,gas-impermeable microfluidic device (13) made from COP, thenOH-Bupropion metabolism may more accurately be predicted.

5. Compound Distribution Kit Validation Experiments

Results from computational models, such as COMSOL Multiphysics (COMSOL),may be compared to results from the compound distribution kit presentedherein in order to validate the effectiveness of the compounddistribution kit. FIG. 92 that shows a COMSOL model can predict theoutlet concentrations of compounds based on parameters obtained fromstatic vial studies. COMSOL models can help inform flow rates and otherexperimental perimeters. Absorption studies may be performed onmaterials, such as polydimethylsiloxane (PDMS), in vials in order tocharacterize those materials. The results from these absorption studieson materials may be input into a computational model of a microfluidicdevice. Computational models can help inform flowrates and otherexperimental parameters.

Once absorption studies are done on particular materials, they may becompared to computation models. FIG. 95 shows a comparison ofcomputational (COMSOL) model flow study results and actual flow studyresults for the small-molecule compound Rhodamine. FIG. 95 shows thatthe flow results fit the COMSOL model for the outlet concentrations ofthe compound. Rhodamine tends to have a lower rate of absorption, buthigher extent of absorption, which can saturate its surroundings overtime. The importance of this is that despite initially seeing hugelosses of Rhodamine, after a period of time, the rate of Rhodamine lossdiminishes significantly.

FIGS. 96A and 96B show a comparison between computational (COMSOL) modelresults and actual experimental results for cellular exposure ranges ofthe small-molecule compound Rhodamine. FIG. 96A shows experimentalresults of the cellular exposure range of the small-molecule compoundRhodamine for a first channel of a microfluidic device. FIG. 96B showscomputational (COMSOL) model results of the cellular exposure range ofthe small-molecule compound Rhodamine for a single channel of amicrofluidic device. The charts in FIGS. 96A and 96B show that thecomputational (COMSOL) model accurately predicted Rhodamine absorptioninto the materials making up microfluidic devices, particularly PDMS.

FIGS. 97A and 97B show a comparison between a computational (COMSOL)model results and actual experimental results for cellular exposureranges of the small-molecule compound Rhodamine. FIG. 96A showsexperimental results of the cellular exposure range of thesmall-molecule compound Rhodamine for a second channel of a microfluidicdevice. FIG. 96B shows computational (COMSOL) model results of thecellular exposure range of the small-molecule compound Rhodamine for asecond channel of a microfluidic device. The charts in FIGS. 97A and 97Bshow that the computational (COMSOL) model accurately predictssmall-molecule absorption into the materials making up microfluidicdevices, particularly PDMS.

FIGS. 98A and 98B show a comparison between a computational (COMSOL)model results and actual experimental results for cellular exposureranges of the small-molecule compound Coumarin. FIG. 98A showsexperimental results of the cellular exposure range of thesmall-molecule compound Coumarin for a first channel of a microfluidicdevice. FIG. 98B shows computational (COMSOL) model results of thecellular exposure range of the small-molecule compound Coumarin for afirst channel of a microfluidic device. It was found that thecomputational (COMSOL) model did not accurately predict the absorption,because the model did not take into account the rest of the flow systemoutside the microfluidic device. For this experiment the microfluidicdevice was in fluidic communication with a perfusion manifold assembly.The compound Coumarin was especially susceptible to absorption into oneof the materials making up the perfusion manifold assembly, SEBS. Assuch, the computational (COMSOL) model did not accurately predict theabsorption into the entire flow system.

FIGS. 99A and 99B show a comparison between a computational (COMSOL)model results and actual experimental results for cellular exposureranges of the small-molecule compound Coumarin. FIG. 99A showsexperimental results of the cellular exposure range of thesmall-molecule compound Coumarin for a second channel of a microfluidicdevice. FIG. 99B shows computational (COMSOL) model results of thecellular exposure range of the small-molecule compound Coumarin for asecond channel of a microfluidic device. It was found that thecomputational (COMSOL) model did not accurately predict the absorption,because the model did not take into account the rest of the flow systemoutside the microfluidic device. For this experiment the microfluidicdevice was in fluidic communication with a perfusion manifold assembly.The compound Coumarin was especially susceptible to absorption into oneof the materials making up the perfusion manifold assembly, SEBS. Assuch, the computational (COMSOL) model did not accurately predict theabsorption into the entire flow system.

FIG. 100 shows experimental results for cellular exposure of thesmall-molecule compound Rhodamine in a two-channel microfluidic devicecomprising a PDMS membrane at a flow rate of 60 uL/hr.

FIG. 101 shows experimental results for cellular exposure of thesmall-molecule compound Rhodamine in a two-channel microfluidic devicecomprising a PDMS membrane without pores at a flow rate of 60 uL/hr.

FIG. 102 shows experimental results for cellular exposure of thesmall-molecule compound Coumarin in a two-channel microfluidic devicecomprising a PDMS membrane at a flow rate of 150 uL/hr.

FIG. 103 shows experimental results for cellular exposure of thesmall-molecule compound Coumarin in a two-channel microfluidic devicecomprising a PDMS membrane without pores.

FIG. 105 shows a timeline for a flow test of two small-moleculecompounds, Drug X and Drug Y. The dose concentration of Drug X was 10 μMand the dose concentration of Drug Y was 1 μM. For the experiment shownin FIG. 106 the end point analysis was liquid chromatography-massspectrometry.

FIGS. 106A and 106B show a summary of flow studies of Drug X in a firstchannel of a two-channel microfluidic device. FIG. 106A shows the outletconcentration of Drug X over time. FIG. 106B shows cellular exposureranges in the first channel. FIGS. 106A and 106B show that Drug X wasabsorbed into the system. The loss of Drug X is consistent with a highlyabsorbing molecule as nearly all the compound is recoverable at 72hours, showing that the microfluidic device material became saturated.FIGS. 106A and 1068 show that over time cell exposure to Drug X would bebetween 80-100%. The media carrying Drug X in FIGS. 106A and 106B alsocontained 2% fetal bovine serum (FBS).

FIGS. 107A and 107B show a summary of flow studies of Drug X in a secondchannel of a two-channel microfluidic device. FIG. 107A shows the outletconcentration of Drug X over time. FIG. 107B shows cellular exposureranges in the first channel. FIGS. 107A and 107B show that Drug X wasabsorbed into the system. The second channel flow rate may possibly beincreased in order to lessen compound absorption.

FIGS. 108A and 108B summarize flow studies of Drug Y in the firstchannel of a microfluidic device. FIG. 108A shows the outletconcentration of Drug Y over time. FIG. 108B shows the range of cellularexposure in the first channel of the microfluidic device over time. Thecompound loss is consistent with a highly absorbing molecule as nearlyall the compound is recovered over 72 hours in the effluent, as thematerial making up the microfluidic device becomes saturated. Over timecellular exposure of Drug Y would be between 80-100%. The media carryingDrug Y in FIGS. 108A and 1088 also contained 2% fetal bovine serum(FBS).

FIGS. 109A and 109B summarize flow studies of Drug Y in the secondchannel of a microfluidic device. FIG. 109A shows the outletconcentration of Drug Y over time. FIG. 109B shows the range of cellularexposure in the second channel of the microfluidic device over time. Thecompound loss in the second channel of the microfluidic device pointstowards absorption. The flow rate may be increased to perhaps decreasecompound absorption.

The compound distribution kit was used successfully to decide whether ornot to commence a drug-study in an Organ-Chip with cells. It wascontemplated to test cannabidiol (CBD oil) in microfluidic devicesseeded with cells (for liver, skin, lung, kidney, etc.) for toxicity,efficacy, and/or ADME. The compound distribution kit was run to assessthe ability at several flow rates. The Compound Distribution Kit foundcomplete/total absorption or loss of compound in the microfluidic devicefabricated from entirely PDMS, which indicated that testing CBD on cellsin PDMS microfluidic devices could most likely not be supported(compound loss was too significant) even at the highest flow rate.Measured outlet concentrations of the compound (CBD) were “0” andnothing could be detected. Decision was made not to pursue testing CBDon a microfluidic device fabricated entirely from PDMS. However, other,low-absorbing embodiments discussed herein would be excellent platformsto test the effects of CBD oil on cells.

FIGS. 115A-D show the results of an experiment testing the absorption ofa compound, herein called Compound Z, in a PDMS microfluidic devicecomprising liver cells using the compound distribution kit. FIG. 115Ashows nearly complete absorption of Compound Z at low flow rates, suchas 30 uL/hr. FIG. 115B shows that significant absorption (nearly 80%loss) of Compound Z at high flow rates, such as 150 uL/hr. FIG. 115Cshows cellular exposure of Compound Z in said first channel of thecompound at 30 uL/hr. FIG. 115D shows cellular exposure of Compound Z insaid first channel of the compound at 150 uL/hr. Experiments were alsorun at a higher concentration to compensate for compound loss. Increaseddosing concentration of Compound Z was conducted and the recoveredoutlet concentration was used as the effective “cellular exposureconcentration.” Increasing the dosing concentration increases thelikelihood of a false positive (compound is not toxic, but a toxiceffect is seen in the microfluidic device), but eliminates thepossibility of a false negative (compound is actually toxic, but themicrofluidic device does not show any toxic response). It is to be notedthat liver cells were used in these experiments, however any cell typeand related readout is contemplated.

Throughout the validation experiments several sources of variabilitywere identified. These sources of variability may be targeted in orderto decrease the total variability in the compound distribution kit.Variability may arise from differences between culture modules overtime, including but not limited to the formation of bubbles. Variabilitymay also arise from user inconsistencies, such as dosing concentrationissues (precipitation, weighing error, dilution error, etc.), notaspirating perfusion manifold assembly outlet reservoirs between timepoints resulting in sample pooling, not aspirating perfusion manifoldassembly reservoirs at the start of the experiment after the ignitionflush resulting in sample dilution, pipetting errors, protocoldeviation, etc. Variability may also arise from material equivalency,such as microfluidic devices fabricated from PDMS versus microfluidicdevices fabricated from other polymers, or microfluidic devices thathave or have not been treated. Variability may also arise from theexclusion of certain components in order to ease use of the compounddistribution kit. For example, when using the compound distribution kiton microfluidic devices for use with testing cells, the cells may beexcluded. However, the exclusion of cells may give rise to a slightvariability.

6. Reciprocation Experiments

Experiments were run to see if reciprocating media through a perfusionmanifold assembly to both COP and PDMS microfluidic devices comprisingliver cells would improve liver recapitulation. Hepatocyte albuminproduction was measured as a readout of liver cell health. Any cell typeis contemplated, however liver cells were chosen to be used.

FIG. 112 shows a graph of albumin production in a PDMS and COPmicrofluidic devices comprising liver cells before and afterreciprocating fluid. It may be seen in FIG. 112 that reciprocating fluidleads to an increase in albumin production as compared to single passflow.

The results shown in FIG. 112 were surprising and completely unexpected.The expectation was that the rates of albumin production would beconserved, and would not decline as this would indicate decline ofhepatocyte function. Increased albumin production rate indicates anincrease in metabolic function. It was desired to confirm theunderstanding that rapid reciprocation leads to an increase in albuminproduction. To do this, the scientists: repeated the experimental planof used to achieve the data shown in FIG. 112, hoping to replicate theresults/albumin trend, took additional albumin samples after returningthe microfluidic devices to single-pass/uni-directional flow (afterreciprocating for 24 hrs). If the results shown in FIG. 112 were valid,the results of the following experiment would predict a similar increasein albumin production after reciprocating microfluidic devices for 24hrs as was done in the prior experiment, and possibly see a return tolower albumin production levels after returning microfluidic devices tosingle-pass flow.

FIG. 113 shows albumin production in PDMS microfluidic devicescomprising liver cells before and after reciprocating fluid. The resultsof FIG. 113 confirm linkage between reciprocation protocol and increasedalbumin production and indicate reversibility of the phenomenon.

Based on the data shown in FIGS. 112 and 113, reciprocation was seen toimprove albumin production in both COP and PDMS microfluidic devices.Furthermore, albumin production was at physiologically relevant levelsin both the COP and PDMS microfluidic devices following the use ofreciprocation.

7. Gas-Permeable Microfluidic Device Gas-Control Using IncubatorExperiments

As was previously described, gas concentrations within microfluidicdevices may be controlled using gas-control incubators. It is of note,that the experiments described below are related to entirelygas-permeable microfluidic devices (12) fabricated from gas-permeablematerials, such as the microfluidic device of U.S. Pat. No. 8,647,861.

Of the various gases that cells are exposed to, oxygen, or lack thereof,is responsible for many fundamental cellular properties and processes.FIG. 116 shows a diagram of oxygen tensions in various human organs.Oxygen, carbon dioxide, and various gases are known to influence thebiological function of cells and can have a profound effect in tissuesand various disease states. For example, oxygen tension differsdramatically in the human body across organs, yet traditional cellculture techniques do not take this into account.

To modify the oxygen microenvironment in gas-permeable microfluidicdevices (12), a gas-controlled incubator may be set to the desiredoxygen setpoint and a desired cell culture protocol may be followed.FIG. 117 shows a diagram of gas exchange in a gas-permeable microfluidicdevice (12). Per FIG. 117, the method of gas transport in thegas-permeable microfluidic device (12) includes gas exchange between anincubator and the microfluidic device material, the microfluidic devicematerial and the cell culture media, and the cell culture media and thecells (33). When a gas-permeable microfluidic device (12) isequilibrated to the incubator oxygen, a first (3) and a second (4)channel may be considered experience equivalent oxygen concentrations.Additionally, when using highly permeable microfluidic devices (12),such as ones fabricated from silicone, inlet media oxygen concentrationsin perfusion manifold assembly reservoirs and flow rate will notsignificantly influence the oxygen microenvironment in the gas-permeablemicrofluidic device (12). Note that the addition of cells (33) andmicrobes (36) will change the channel oxygen concentrationsindependently based on cellular oxygen consumption.

With regards to instrumentation, several exemplary pieces of equipmentwere found through experimentation. The Thermo Scientific™ Heracell™240i was found to be the best gas-control incubator for reliability andefficiency. It was found in general that any standard cell cultureincubator may be used with a separate gas controller. The BioSphereixProOx 360 was found to be the best gas controller, which injectsnitrogen to displace oxygen within the incubator, being regulated by anoxygen sensor placed inside the incubator.

To begin experiments the incubators are at atmospheric conditions.Inducing hypoxia in the incubator, and thus the gas-permeablemicrofluidic devices (12), perfusion manifold assemblies (14), andculture modules (42) may take a significant amount of time as may beseen in FIG. 118. FIG. 118 shows a diagram of the results ofgas-permeable microfluidic device (12) response to various oxygen phaseswhile in a cell culture incubator. Oxygen measurements were taken of agas-permeable microfluidic device (12) outlet under flow at 30 μL/hrflow in a culture module, wherein the flow is with 18.5% oxygen into theinlet. As seen in FIG. 118 the incubator starts at atmospheric oxygenlevels (18.5% in a humidified incubator), reaches 1% oxygen setpoint(seen with a long tail-end), and returns to atmospheric oxygen upon theincubator being opened to the atmosphere.

Once equilibrium is achieved in a gas-permeable microfluidic device(12), first (3) and second (4) channel gas concentrations will maintainthe incubator oxygen setpoint when flowing fluid or media if themicrofluidic device (12) is fabricated out of a high permeabilitymicrofluidic device material. Thus, the inlet fluid or mediaconcentrations are largely inconsequential if the microfluidic device ishighly permeable. This point was proven during experimentation, as seenin FIG. 119. FIG. 119 shows a diagram of the results experimental oxygenmeasurements of microfluidic device outlets under water flow at 100μL/hr in a culture module with either 18.5% oxygen (oxygenated), or 1-5%oxygen (hypoxic) concentrations, in a 1% oxygen incubator. Thegas-permeable microfluidic device (12) and system were equilibrated tothe incubator environment for 12 hours prior. When flowing fullyoxygenated water or hypoxic (1-5% oxygen) water at 100 uL/hr, first (3)and second (4) channel oxygen outputs reach below 1.5-2% oxygen withinminutes. The experiment was also simulated and confirmed in athree-dimensional gas-permeable microfluidic device model using thefinite element analysis software COMSOL Multiphysics as seen in FIG.120. FIG. 120 shows a diagram of the results of a COMSOL Multiphysicssimulation plot of a PDMS microfluidic device first channel and secondchannel volume averages of the same conditions with oxygenated media.Therefore, it may be seen that controlling the gas-concentration insidean entirely gas-permeable microfluidic device (12) using a gas-controlincubator is highly effective.

Furthermore, flow rates below 1000 μL/hr minorly contribute to channeloxygen concentration because of the high diffusion rate of highlypermeable materials making up these microfluidic devices (12) and theincubator itself. Oxygen diffuses out of the fluid or medium much fasterthan the oxygen being replaced in the flowing medium. FIG. 121 shows adiagram of results of a COMSOL Multiphysics simulation plot of PDMSmicrofluidic device first and second channel volume averages for 30μL/hr and 1000 μL/hr flow rates with oxygenated inlet water in a 1%oxygen incubator. It may be seen in FIG. 121 that flow rate is not asubstantial variable in controlling the gas environment of agas-permeable microfluidic device (12) within a culture module (42).

Additionally, high flow rates are less practical since it will requirereplenishing fluid reservoirs, such as fluid reservoirs (19) in FIG. 7,which involves opening the incubator door and resetting oxygen levelswithin the incubator. FIG. 118 shows the affect of opening the incubatordoor on the oxygen levels within the gas-permeable microfluidic device(12). When the incubator environment is disturbed, such as opening thedoor to change flow rate, access the microfluidic devices, accessanother experiment, etc., microfluidic device equilibration will be influx. Since the diffusion of oxygen in the gas-permeable microfluidicdevices (12) occurs in minutes, channels (3, 4) will re-equilibratewhile the incubator oxygen concentration rises and reduces back to thesetpoint. Quick door openings may only cause small oxygen rises inanaerobic incubators and a relatively short microfluidic device recoverytime (in the range of a few hours): a five second door opening willresult in an additional 1.5 hours to reach gas-permeable microfluidicdevice (12) oxygen concentrations below 2%, as seen in FIG. 122. FIG.122 shows a diagram of results of recovery time when opening anincubator door. Oxygen measurements were taken at the outlet of amicrofluidic device under 100 μL/hr water flow in a culture moduleinside an incubator set to 1% oxygen. The microfluidic device, culturemodule, and remainder of system were equilibrated to the incubatorenvironment for 12 hours prior. The incubator door was opened for fiveseconds before starting measurements. The oxygen recovery time largelydepends on the incubator and gas control system, as large single-dooredincubators will be less efficient than multi-doored or high nitrogenpressure input systems.

Handling gas-permeable microfluidic devices outside a hypoxic incubatorand perfusion manifold assembly (14) should be performed as quickly aspossible during low-oxygen experiments on gas-permeable microfluidicdevices. Only being able to access gas-permeable microfluidic devices(12) during low-oxygen experiments for very short periods of time mayimpact protocol steps that require direct access to a microfluidicdevice, such as inoculating microfluidic devices with bacteria. COMSOLsimulations indicate oxygen concentrations will continuously doublewithin minutes and reach atmospheric oxygen within 30 minutes as seen inFIG. 123. FIG. 123 shows a diagram of results of a COMSOL Multiphysicssimulation plot of PDMS microfluidic device (12) first (3) and second(4) channel volume averages of a static PDMS microfluidic device (12)equilibrated to 1% oxygen and exposed to atmospheric oxygen.Experimental results concluded an oxygen half-life of around 6 minutesfor the gas-permeable microfluidic device (12) outside the culturemodule (42) and perfusion manifold assembly (14). After five half-lives,steady-state is considered reached (97% of steady-state) which equatesto around 30 minutes, confirming the COMSOL simulation.

Experimental timing was found for the present system, includinggas-permeable microfluidic devices (12), perfusion manifold assemblies(14), and culture modules (42). Cell culture incubators were found totake 2-5 hours to reach low or anaerobic oxygen levels. Gas-permeablemicrofluidic devices (12) were found to reach low or anaerobic oxygenequilibration in 3 hours when in the incubator with connection toperfusion manifold assemblies (14) and culture modules (42), wherein thehalf-life of oxygen was found to be 35 minutes for the gas-permeablemicrofluidic devices (12) in that experimental setup. Gas-permeablemicrofluidic devices (12) were found to reach low or anaerobic oxygenequilibration in 30 minutes when in the incubator without contact toperfusion manifold assemblies (14) and culture modules (42), wherein thehalf-life of oxygen was found to be 6 minutes for the gas-permeablemicrofluidic devices (12) alone in the incubator.

Cellular oxygen consumption can be a significant contributor to thedepletion of total oxygen within the gas-permeable microfluidic device(12). When considering highly metabolic cells such as colonic epithelialcells characterized by an oxygen uptake rate of 2020 nmol/hr, channeloxygen levels differ under standard oxygenated cell culturingconditions. Using COMSOL, the average top and bottom channel oxygenconcentrations reach 14% and 12% respectively, as seen in FIG. 124. FIG.124 shows a diagram of results of a COMSOL Multiphysics simulation plotof PDMS microfluidic device first and second channel volume averages ofa microfluidic device with seeded Caco-2 cells in culture conditions or18.5% oxygen incubator and 18.5% oxygen inlet water at 100 μL/hr waterflow rate. However, a local microgradient is also formed where oxygenconcentrations decrease close to the cell layer, reaching as low as 2%oxygen right at the center of the cell layer as seen in FIG. 125. FIG.125 shows a diagram of a PDMS microfluidic device oxygenmicroenvironment with the addition of Caco-2 cells. FIG. 125 shows across-sectional surface pot of water oxygen concentrations in the centerof the microfluidic device. The simulation which produced the resultsshown in FIGS. 124 and 125 highlights the importance of consideringcellular oxygen uptake and release when designing experiments.

The above study demonstrates the gas-permeable microfluidic device gasenvironment can be easily modified with a culture module placed inside agas-controlled incubator. Other applications include high oxygenenvironments (hyperoxia) or introducing various gasotransmitters. Note,first and second channels are difficult to be controlled independently,the whole microfluidic devices experience the same gas composition ifcell metabolism is not considered. Cell metabolism will significantlycontribute to the gas microenvironment and even introduce local gasgradients. Additional endpoints and controls should be considered whenperforming gas-controlled experiments, such as incorporating hypoxiastains for low oxygen conditions.

1-66. (canceled)
 67. A method of analyzing compound distribution in asystem, comprising: a) providing a system and a first experimentalprotocol for use with said system, said first experimental protocolcomprising introducing a compound into said system and taking actions atone or more timepoints; b) modifying said first experimental protocol togenerate a first modified experimental protocol, wherein said firstmodified experimental protocol comprises measuring compoundconcentration at one or more of said timepoints from said firstexperimental protocol; c) performing said first modified experimentalprotocol; and d) using said measurement of concentration of saidcompound to analyze compound distribution across said system.
 68. Themethod of claim 67, further comprising the step of e) performing saidfirst experimental protocol.
 69. The method of claim 67, wherein saidsystem comprises one or more microfluidic devices.
 70. The method ofclaim 67, wherein said system comprises infusion tubing.
 71. The methodof claim 67, wherein said system comprises syringes.
 72. The method ofclaim 67, wherein said system comprises one or more biological elementsand said first experimental protocol is modified to exclude at least oneof said one or more biological elements.
 73. The method of claim 72,wherein said first experimental protocol comprises compound testing onsaid biological elements.
 74. The method of claim 67, wherein said firstexperimental protocol comprises cells and said first modifiedexperimental protocol does not comprise cells.
 75. The method of claim67, wherein said system comprises coatings and said first experimentalprotocol is modified by excluding coatings.
 76. The method of claim 67,wherein said first modified experimental protocol does not comprisetaking actions at one or more timepoints of said first experimentalprotocol.
 77. The method of claim 67, wherein said performing ameasurement of the concentration replaces said taking actions at one ormore timepoints.
 78. The method of claim 67, wherein said first modifiedexperimental protocol is modified in that only a subset of inputcompound concentrations is included in said modified experimentalprotocol as compared to said first experimental protocol.
 79. The methodof claim 67, wherein said first modified experimental protocol in thatporous elements are excluded as compared to said first experimentalprotocol.
 80. The method of claim 67, wherein said system includes afirst microfluidic device comprising a first membrane with pores. 81.The method of claim 79, wherein said system is replaced with a secondsystem in said modified experimental protocol, said second systemincluding a second microfluidic device not comprising a membrane withoutpores in at least one region in which said first membrane comprisespores.
 82. The method of claim 67, wherein said first experimentalprotocol comprises flowing fluid in said system.
 83. The method of claim82, wherein said system comprises an input port configured to permitfluid input to the system.
 84. The method of claim 82, wherein thesystem comprises an output port configured to permit fluid output fromthe system.
 85. The method of claim 83, wherein said first experimentalprotocol comprises flowing into said input port.
 86. The method of claim84, wherein said first experimental protocol comprises collecting afirst sample from said output port.
 87. The method of claim 84, whereinsaid measuring of the concentration of said compound comprisescollecting a sample from said output port and quantifying saidconcentration of said compound in said sample.
 88. The method of claim67, wherein said first modified experimental protocol further quantifiesthe percentage of said compound that is absorbed into said system. 89.The method of claim 67, further comprising introducing fluid flow tosaid system.
 90. The method of claim 89, wherein said taking actionscomprises sampling effluent.
 91. The method of claim 90, wherein saidfirst experimental protocol further comprises assaying said effluent toachieve an apparent metabolite value.
 92. The method of claim 91,further comprising using said measurement of concentration of saidcompound to correct said apparent metabolite value.
 93. The method ofclaim 91, further comprising using said measurement of concentration ofsaid compound to determine variability of said apparent metabolitevalue.
 94. The method of claim 67, further comprising using saidmeasurement of concentration to determine whether to perform said firstexperimental protocol.
 95. The method of claim 67, further comprising(i) using said measurement of concentration of said compound to generatea second modified experimental protocol; and (ii) performing said secondmodified experimental protocol.
 96. The method of claim 67, wherein saidfirst experimental protocol comprises living cells. 97-105. (canceled)106. A method of determining compound distribution in a system,comprising: a) providing a system and an experimental protocol for saidsystem comprising one or more biological elements; wherein said one ormore biological elements are contacted by a compound; b) modifying saidexperimental protocol by excluding at least one of said one or morebiological elements; c) performing said modified experimental protocol;and d) determining the distribution of said compound in said systemusing by measuring the concentration of said compound in said system.107. The method of claim 106, wherein said experimental protocolcomprises introducing fluid flow into said system.
 108. The method ofclaim 107, wherein said experimental protocol comprises collectingeffluent.
 109. The method of claim 105, wherein said experimentalprotocol comprises assaying said effluent.
 110. The method of claim 106,wherein said biological elements comprise cells.
 111. The method ofclaim 106, wherein said biological elements comprise biologicalcoatings.
 112. The method of claim 106, wherein said system comprisesone or more microfluidic devices.
 113. The method of claim 106, whereinsaid distribution of said compound is used to calculate error bars forresults from said experimental protocol.
 114. The method of claim 106,said percent distribution of said compound is used to calculate halfmaximal inhibitory concentration (IC₅₀) for said experimental protocol.115. A method of assessing compound distribution in a system,comprising: a) providing a system and a first experimental protocol forsaid system, said first experimental protocol comprising introducing acompound into said system; b) modifying said first experimental protocolto generate a modified experimental protocol, said modified experimentalprotocol comprising: i) introducing said compound using a firstconcentration; and ii) performing a first measurement of theconcentration of said compound; c) performing said modified experimentalprotocol; d) comparing said measurement of the concentration of saidcompound to a threshold; and e) performing said first experimentalprotocol if said measurement of concentration surpasses said threshold.116. The method of claim 115, wherein said first experimental protocolfurther comprises introducing fluid flow into said system.
 117. Themethod of claim 116, wherein said first experimental protocol comprisescollecting effluent at one or more time points.
 118. The method of claim117, wherein said first experimental protocol comprises assaying saideffluent.
 119. The method of claim 115, wherein said biological elementscomprise cells.
 120. The method of claim 115, wherein said biologicalelements comprise biological coatings.
 121. The method of claim 115,wherein said system comprises one or more microfluidic devices.
 122. Themethod of claim 117, wherein said first measurement is performed atleast one of said one or more timepoints of said first experimentalprotocol.
 123. The method of claim 115, wherein said measurement of theconcentration of said compound to a threshold are compared by dividingsaid first measurement by said first concentration to obtain a firstratio.
 124. The method of claim 123, wherein the said threshold is afirst ratio value above one of 10%, 20%, 33%, 50%, 66%, and 75%. 125.The method of claim 115, wherein said modified experimental protocolfurther comprises measuring an input compound concentration, and whereinthe said first measurement is divided by the measured said inputconcentration to obtain a measured ratio. 126-293. (canceled)